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Ryanodine Receptor Calcium Release Channels
MICHAEL FILL AND JULIO A. COPELLO
Department of Physiology, Loyola University Chicago, Maywood, Illinois
I. Introduction 894II. History 894
III. Structure-Function Overview 895IV. Excitation-Contraction Coupling 897
A. DHPR-RyR interaction 897 V. Study of Intracellular Calcium Release 899
VI. Study of Single Ryanodine Receptor Channels 900 VII. Permeation Properties 901
VIII. Action of Ryanodine 902IX. Overview of Ryanodine Receptor Regulation 902 A. Cytosolic Ca 2 903B. Luminal Ca 2 903C. ATP and Mg2 903D. Redox status 903E. Cyclic ADP-ribose 904F. Phosphorylation/dephosphorylation 904
X. Regulation by Closely Associated Proteins 904 A. Calmodulin 904B. Calsequestrin 904C. FK-506 binding protein 905D. DHPR-loop peptide 905
XI. Calcium Regulation Paradox 906XII. Steady-State Calcium Dependence 907
XIII. Calcium Activation Kinetics 907 A. Rate of Ca 2 activation 907B. Rate of Ca 2 deactivation 908
XIV. Feedback Calcium Regulation 908XV. Potential Negative Control Mechanisms 908
A. Ca 2-dependent inactivation 909B. Stochastic attrition 909C. Adaptation 909D. Activation-dependent or “fateful” inactivation 910E. Depletion/luminal Ca 2 effects 910F. Allosteric or coupled gating 910
XVI. Adaptation Debate 911 A. DM-nitrophen Ca 2 overshoot 911B. Ca 2 overshoot concern 911C. Differing interpretations 912
XVII. Modal Gating 913XVIII. Concluding Remarks 914
Fill, Michael, and Julio A. Copello. Ryanodine Receptor Calcium Release Channels. Physiol Rev 82: 893–922,2002; 10.1152.physrev.00013.2002.—The ryanodine receptors (RyRs) are a family of Ca 2 release channels found onintracellular Ca 2 storage/release organelles. The RyR channels are ubiquitously expressed in many types of cellsand participate in a variety of important Ca 2 signaling phenomena (neurotransmission, secretion, etc.). In striatedmuscle, the RyR channels represent the primary pathway for Ca 2 release during the excitation-contraction coupling process. In general, the signals that activate the RyR channels are known (e.g., sarcolemmal Ca 2 influx or depolarization), but the specific mechanisms involved are still being debated. The signals that modulate and/or turnoff the RyR channels remain ambiguous and the mechanisms involved unclear. Over the last decade, studies of
Physiol Rev
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RyR-mediated Ca 2 release have taken many forms and have steadily advanced our knowledge. This robust field,
however, is not without controversial ideas and contradictory results. Controversies surrounding the complex Ca 2
regulation of single RyR channels receive particular attention here. In addition, a large body of information is
synthesized into a focused perspective of single RyR channel function. The present status of the single RyR channel
field and its likely future directions are also discussed.
I. INTRODUCTION
Calcium is a common second messenger (38, 42, 123)
that regulates many processes in cells (e.g., contraction,
secretion, synaptic transmission, fertilization, nuclear
pore regulation, transcription). Although cells are typi-
cally bathed in solutions containing a relatively high Ca 2
concentration (i.e., in the millimolar range), the cyto-
plasm of most cells contains much lower resting Ca 2
concentrations (i.e., in the 100 nM range). Thus Ca 2
entry across the surface membrane can substantially ele-
vate cytosolic Ca 2 levels providing the Ca 2 trigger sig-
nals for a large number of physiological processes. Thesurface membrane, however, is not the only source of
triggering Ca 2 signals. Most cells have developed an
additional pathway to generate localized and fast trigger
Ca 2 signals deep inside the cell. This other pathway
involves specialized intracellular Ca 2 storage/release or-
ganelles.
Cells have many different types of Ca 2 transporters
(channels, exchangers, pumps) that modulate cytosolic
Ca 2 levels. The rich array of Ca 2 transport proteins in
the surface and intracellular membranes provides numer-
ous potential Ca 2 signaling pathways that can respond to
many different types of stimuli. Specificity of any oneCa 2 signal pathway to a particular process is often made
possible by assembling the particular signaling proteins
into a local complex. Localizing the Ca 2 signaling effec-
tively allows Ca 2 to be effectively directed to a particular
physiological function. Certain vascular smooth muscles
are a good example of this. In these smooth muscles, large
global intracellular Ca 2 signals activate the contractile
machinery causing the cell to contract. However, small
very localized Ca 2 signals (i.e., Ca 2 sparks) that arise
from intracellular Ca 2 stores activate certain K chan-
nels in the surface membrane promoting relaxation of the
cell (219).
The primary intracellular Ca 2 storage/release or-ganelle in most cells is the endoplasmic reticulum (ER).
In striated muscles, it is the sarcoplasmic reticulum (SR).
The ER and SR contain specialized Ca 2 release channels.
There are two families of Ca 2 release channels: the
ryanodine receptors (RyRs) and inositol trisphosphate
receptors (IP3Rs). Each family of Ca 2 release channels
appears to contain three different isoforms (300, 309).
However, there may be a fourth type of RyR channel in
fish (i.e., RyR-slow; Refs. 90, 210). The Ca 2 release chan-
nels are large oligomeric structures formed by association
of either four RyR proteins or four IP3R proteins. The RyR
and IP3R proteins share significant homology, and this
homology is most marked in the sequences that may form
the channel’s pore (204, 271, 299, 315a, 367).
Calcium regulates all the RyR and IP3R channels.
However, each Ca 2 release channel has its own distin-
guishing functional attributes. The IP3R channels require
the presence of inositol 1,4,5-trisphosphate. The activity
of one type of RyR channel is coupled to a “ voltage
sensor ” in the plasma membrane. The Ca 2-induced Ca 2
release process governs the activity of the other types of
RyR channel. The distinguishing functional attributes of
each RyR or IP3R channels likely underlie the spatiotem- poral complexity of intracellular Ca 2 signaling in cells.
During the last 10 years, there have been numerous
reviews on several aspects of regulation of RyR-mediated
Ca 2 signaling and/or related topics (e.g., Refs. 18, 43, 60,
69, 76, 84, 87, 92, 112, 179, 195, 199, 202, 225, 245, 252, 253,
255, 260, 265, 281, 287, 300, 301, 307, 315a, 335, 346, 354).
Here, we focus on the RyR Ca 2 release channels found in
striated muscle. The controversial and complex Ca 2 reg-
ulation of single RyR channels is given particular atten-
tion. An attempt has been made to cover this large rapidly
growing and dynamic body of information. Regrettably, it
is impossible to describe all of the quality studies that
characterize this field. It is hoped that the single-channel
perspective of this review will be useful to readers seek-
ing to understand the complex dynamics of single RyR
channel regulation
II. HISTORY
The significance of SR Ca 2 release during skeletal
muscle contraction was recognized many decades ago
(for review, see Refs. 38, 42, 260). Electrophysiological
experiments and 45Ca autoradiography studies provided
evidence in intact muscle that Ca 2 is released from the
terminal cisternae of the SR in response to depolarizationof surface membrane invaginations called transverse tu-
bules (t tubules) (reviewed in Ref. 260). A number of
studies followed, and a putative mechanism was pro-
posed (43, 251–255, 265–267, 296), namely, that there is a
physical coupling between an integral t-tubule protein
and the SR Ca 2 release channel. The idea was that
voltage-dependent movements of charged particles in the
integral t-tubule protein activate the SR Ca 2 release
channel through this physical coupling. This mechanism
is generally referred to as depolarization-induced Ca 2
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release (DICR). Parallel work in other experimental prep-
arations (i.e., SR vesicles and skinned muscle fibers) con-
firmed the existence and importance of the DICR mech-
anism in skeletal muscle (38, 94, 121, 127, 138, 150, 151,
183, 260, 296, 321).
In other early works, it was realized that a small
increase in cytosolic Ca 2
concentration could inducemassive intracellular SR Ca 2 release events in both skel-
etal and cardiac muscle (75, 89). This phenomenon is
often called Ca 2-induced Ca 2 release (CICR). Later,
meticulous studies by Fabiato (80, 81) established that the
CICR process was sensitive to both the speed and ampli-
tude of the applied Ca 2 trigger. These studies and others
helped reveal the complexity of the regulatory mecha-
nisms that control SR Ca 2 release.
A major advancement was the identification of the SR
Ca 2 release channel using an alkaloid called ryanodine.
Ryanodine is found naturally in the stem and roots of the
plant Ryania speciosa. The ryanodine alkaloid was first
isolated as a potential insecticide (73, 133), and its potent
paralytic action on skeletal and cardiac muscles was im-
mediately evident (73, 82, 93, 218, 311, 312). However, its
action was dif ficult to understand. Specifically, it was
dif ficult to understand how ryanodine could induce rigid
paralysis in skeletal muscle but flaccid paralysis in car-
diac muscle.
The ryanodine alkaloid was shown to inhibit SR Ca 2
release by binding with high af finity to a protein present in
the SR membrane (41, 80, 88, 93, 309 –312, 345, 347). The
ryanodine binding protein was purified (39, 88, 124, 147,
240, 241) and existed in a tetrameric complex (149) whose
shape was subsequently visualized using electron micros-copy (131, 147). Interestingly, the size and shape of the
ryanodine binding protein complex was similar to that of
the “feet” structures, which appear to physically link the t
tubule and SR membranes (83, 94). Incorporation of the
ryanodine binding protein complex into artificial planar
lipid bilayers revealed that this structure was an ion chan-
nel (124, 130, 147, 294). The RyR channel is a poorly
selective Ca 2 channel (selectivity Ca 2 /K 6) with very
high conductance (700 pS when K is charge carrier
and 100 pS when Ca 2 is charge carrier). The channel is
regulated by Ca 2, Mg2, ATP, and caffeine. Interestingly,
application of the ryanodine alkaloid locks the channel in
a slow-gating subconductance state (124, 147, 294, 337).Openings of the ryanodine-bound channel are long-lived
and have a smaller unit current (1/3 or 1/2 of control
amplitude). This action of ryanodine on single RyR chan-
nel function is quite distinctive and is now often used to
functionally identify the channel.
These early single-channel studies unambiguously
identified the RyR channel as the SR Ca 2 release channel
in striated muscle (84, 87). It also became evident how the
ryanodine alkaloid can induce rigid paralysis in skeletal
muscle but flaccid paralysis in cardiac muscle. In cardiac
muscle, ryanodine promotes Ca 2 leak from intracellular
stores because it locks the RyR channels in the long-lived
subconduction state. This “leaked” Ca 2 is rapidly re-
moved from the cell by the relatively strong surface mem-
brane Ca 2 extrusion mechanisms present in cardiac
muscle (17, 20). Intracellular Ca 2 stores eventually de-
plete, yielding the observed flaccid cardiac paralysis. Incontrast, skeletal muscle lacks the strong surface mem-
brane Ca 2 extrusion mechanisms present in cardiac
muscle. The ryanodine-induced Ca 2 leak from intracel-
lular stores causes Ca 2 to accumulate in the cytoplasm.
The result is abnormally high cytoplasmic Ca 2 levels that
induce sustained contraction (i.e., the rigid skeletal mus-
cle paralysis).
III. STRUCTURE-FUNCTION OVERVIEW
Molecular cloning studies have defined three differ-
ent RyR isoforms in fish, amphibian, birds, and mammals
(6, 60, 205, 215, 225, 245, 301, 307, 315a). Although the RyR
proteins share significant homology with the IP3R recep-
tors, they have little homology with the more widely
studied voltage-dependent Ca 2 channels found in the
surface membrane (204, 315a, 321). In mammals, the three
RyR isoforms (RyR1, RyR2, and RyR3) are encoded by
three different genes on different chromosomes (186, 205,
318, 315a, 369). The RyR proteins are in a variety of
tissues, but the highest densities are in striated muscles
(5, 6, 148, 205, 212, 225, 227, 231, 233, 299, 308). There
may also be structurally, and perhaps functionally, dis-
tinct splice variants of these channels (102, 221, 370).However, the functional significance and distribution
among fiber types of RyR splice variants are currently
unknown.
In mammalian striated muscles, the expression of the
different RyR protein isoforms is tissue specific. The pre-
dominant RyR isoform in skeletal muscle is the RyR1
protein, commonly referred to as the skeletal RyR isoform
(60, 195, 225, 245, 299). The RyR2 protein is the most
abundant isoform in cardiac muscle, and the RyR2 protein
is commonly referred to as the cardiac RyR isoform. The
RyR3 protein is also found in mammalian striated mus-
cles, but at relatively low levels (98, 308, 325). For exam-
ple, the RyR3 protein represents 5% of the overall RyR population in diaphragm (135, 213).
What is the physiological role of the RyR3 channel in
striated muscle or elsewhere? Mice missing the RyR1 and
RyR2 gene products (i.e., RyR1 or RyR2 knock-outs) die
early during embryonic development (317, 320). In con-
trast, mice missing the RyR3 gene product (i.e., RyR3
knock-out) lead relatively normal lives and have quite
normal striated muscles (55, 317). Interestingly, the CICR
process in neonate skeletal muscle fibers from RyR3-
knockout mice appears to be impaired (357). The impli-
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cations of this observation are not yet clear. In any event,
it appears that the physiological role of the RyR3 channel
in mammals, unlike that of the RyR1 and RyR2 channels,
can be readily compensated for both during development
and in the adult.
The RyR nomenclature and distribution are some-
what different in nonmammalian striated muscle (35, 148,212, 232, 307). Nonmammalian skeletal muscle (e.g., frog,
fish, and chicken) contains nearly equal amounts of two
different RyR proteins, named the -RyR and -RyR.
These isoforms are homologs of mammalian RyR1 and
RyR3 forms, respectively (5, 6, 148, 212, 223, 227, 233, 308,
307). It is known that embryonic chicken skeletal muscle
cells fail to develop normal excitation-contraction cou-
pling in the absence of -RyR (132), the RyR1 analog.
Although there have been no -RyR knock-out studies, all
indications are that the -RyR must play some fundamen-
tally important role in the operation of nonmammalian
skeletal muscle (307). One likely possibility is that the
-RyR isoform participates in the CICR process, effec-
tively amplifying the initial DICR mediated by the -RyR
isoform.
The RyR proteins are also expressed in smooth mus-
cle and many nonmuscle tissues (e.g., neurons). These
other tissues generally contain multiple RyR isoforms.
For example, expression of all three RyR isoforms has
been reported in aortic smooth muscle (162, 186), cere-
brum (101, 115, 220), and cerebellum (101, 115, 187).
These tissues also express multiple IP3R isoforms, and
thus one cell may contain many types of RyR and IP 3R
channels. The significance of this remarkable redundancy
in intracellular Ca 2
release channel expression in non-muscle systems is not clearly understood. One might
speculate that the rich morphological diversity implies
equally rich functional heterogeneity and that the multiple
functionally distinct Ca 2 release channels may govern
different Ca 2 signaling tasks. Consequently, Ca 2 release
channel diversity may represent one factor that allows
cells to simultaneously carry out the myriad of Ca 2
signaling tasks they need to do to survive. Even striated
muscles, which are highly specialized for the Ca 2 signal-
ing that governs contraction, must carry out other types
of Ca 2 signaling tasks to survive. Perhaps the RyR3
channel or the low density of IP3Rs expressed in striated
muscles (239, 276) carry out this “other ” Ca 2 signaling. At the amino acid level, the three mammalian RyR
isoforms share 70% identity (115, 215, 318, 315a). Anal-
ysis of the primary amino acid sequences suggests that
the membrane-spanning domains of the RyR are clustered
near its COOH terminus (271, 299, 315a, 367). Truncation
mutants, containing only these putative transmembrane
regions (TMRs), are suf ficient to form Ca 2-selective
channels when incorporated into planar lipid bilayers (23,
24). Mutation of certain regions in and around the puta-
tive TMRs generates channels with altered ion selectivity
(23, 103, 367). Thus there is reasonable certainty that this
region of the protein contains the determinants that form
the Ca 2-selective pore of the RyR channel.
Analysis of the RyR’s primary amino acid sequence
also has revealed several consensus ligand binding (e.g.,
ATP, Ca 2
, caffeine, and calmodulin) and phosphoryla-tion motifs. Experimental verification of these sequences
as potential regulatory sites (e.g., Refs. 23, 45, 47) is
ongoing. For example, substitution of alanine-3882 by a
glutamate (E3882A) in the RyR3 protein results in RyR3
channels with altered Ca 2 sensitivity (45). This implies
that this region of the protein may contain the “Ca 2
sensor ” of the channel. A gross deletion of amino acids
183– 4006 in the RyR1 protein results in channels that lack
caffeine sensitivity and do not inactivate at high Ca 2
concentrations (23). Exchange of amino acids 4187– 4628
of RyR1 for the corresponding cardiac RyR2 sequence
results in channels with increased caffeine sensitivity and
altered Ca 2 sensitivity (66, 67).
Two skeletal muscle diseases, malignant hyperther-
mia (MH) and central core disease (CCD), have also
provided useful insights into the RyR1 structure-function
relationship (180, 366). These diseases are marked by
pathologies like hypercarbia, rhabdomyolisis, and gener-
alized muscle contraction. A defect in the RyR1 channel
function was suspected to be involved in these patholo-
gies (85, 277, 338). It is now known that specific point
mutations in the RyR1 protein are associated with the MH
and CCD diseases (180, 243, 277, 366). Recent reports
describe more than 20 different point mutations in the
human RyR1 isoform that are associated with the MHand/or CCD syndromes (140, 193). The majority of the
mutations are clustered in two regions of RyR1 sequence,
residues 35– 614 (near the NH2 terminal) and 2163–2458 (a
central location). These data are important because these
mutations may reveal important points in the RyR1 struc-
ture that govern its function.
It is clear that the exploration of the RyR structure-
function relationship by mutagenesis is still in its infancy.
This is in stark contrast to the molecular study of other
types of ion channels. There are several reasons why we
have not progressed further or faster in this area. First,
the RyR channels are expressed in intracellular organelles
(ER or SR), and thus single RyR channel function cannotbe directly and easily accessed. For surface membrane
channels, function is often ef ficiently assessed by the
classical patch-clamp method on a few tagged expressing
cells. For the RyR case, function is generally defined in
vitro after the channels have been biochemically isolated
from millions of expressing cells. Second, the RyR is a
huge protein (1/10 the size of a ribosome). This large
size complicates its genetic manipulation as well as the
selection of sites to mutate. Third, recent studies suggest
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that RyR1 channel function may involve inter-RyR domain
interactions (128, 279, 355), and thus some functional
attributes may be controlled by multiple and very com-
plex structural determinants. Despite these and other
technical challenges, future mutagenesis studies promise
to provide some very interesting insights into RyR struc-
ture-function in the not too distant future.Usually, ion channel structure-function studies are
interpreted in the absence of information concerning the
channel’s detailed three-dimensional (3-D) structure.
There are some exceptions where channels or channel
segments have been crystallized. However, the RyR chan-
nel is not one of these exceptions. Intriguingly, some
gross details of the 3-D RyR structure (i.e., general shape)
have been predicted using cryoelectron microscopy and
3-D reconstruction techniques. The RyR1 channel has
fourfold symmetry, presumably reflecting its formation by
four RyR protein monomers (249, 270, 342). The RyR
channel has two distinct domains (249, 270, 342). One is a
large cytoplasmic assembly (29 29 12 nm), consist-
ing of loosely packed protein densities. The other is a
small transmembrane assembly that protrudes (7 nm)
from the center of the cytoplasmic assembly (270, 342).
This transmembrane assembly appears to have a central
hole that can be occluded by a pluglike mass. This hole
and plug may correspond to the Ca 2-selective pore and
its gate (230). In general, the shapes of the RyR1 and RyR2
channels are quite similar (275). There are, however, spe-
cific points of structural heterogeneity. These points may
contribute to the isoform-specific functional attributes of
the RyR channels. Cryoelectron microscopy studies have
also revealed the sites where certain peptides (calmodu-lin, FK binding proteins, and imperatoxin) interact with
the RyR channels (263, 343). Recently, antibodies have
been used to map the location, on the 3-D structure, of a
specific domain (amino acid residues 4425– 4621) that
may be implicated in the Ca 2-dependent regulation of
the RyR channel (15).
Certain structural details of the RyR channel have
also been obtained using certain biophysical approaches.
For example, the width of the RyR2 pore has been esti-
mated from molecular sieving experiments (332). In these
studies, it was found that only cations with a predicted
circular radius smaller than 3.5 Å could permeate through
the channel. In another study, streaming potential exper-iments indicated that the RyR2 channel contains a narrow
region in which H2O (radius 1.5 Å ) and Cs (radius 2 Å )must pass in a single-file fashion (337). Combined, these
results suggest that the RyR2 channel pore is 3 Å wide.
Electrical distance measurements, using trimethylammo-
nium derivatives of varying length, indicated that the
physical length of the voltage drop along the RyR2 chan-
nel pore is 10.4 Å (331). This is consistent with steaming
potential measurements that suggest that three H2O mol-
ecules (9 Å ) occupy the single-file region of the RyR2
channel pore (337). Thus the RyR channels appear to have
a relatively short permeation pathway.
IV. EXCITATION-CONTRACTION COUPLING
A role in striated muscle excitation-contraction (E-C)
coupling is probably the RyR channel’s most notable
claim to fame. There is clear evidence that the RyR chan-
nels interact with voltage-dependent Ca 2 channels (i.e.,
dyhydropyridine receptors; DHPRs) in the nearby t-tubule
membrane (246, 247, 321, 322). This functional interaction
between the DHPR and RyR is commonly referred to as
E-C coupling (Figs. 1 and 2). Depolarization of the t-tubule
membrane (i.e., excitation) induces conformational
changes in DHPR that ultimately lead to activation of RyR
channel in the SR membrane. The activation of RyR chan-
nels leads to massive Ca 2 release from the SR, which in
turn initiates contraction. The bulk of information ad-
dressing RyR channel regulation has been obtained from
studies focused on the process of E-C coupling in striated
muscle.
More than a century ago, Ringer demonstrated that
the cardiac E-C coupling process required the presence of
extracellular Ca 2 (reviewed in Ref. 38). In cardiac mus-
cle, the DHPR receptor (an L-type Ca 2 channel) carries a
small Ca 2 influx that activates the RyR2 channel (16, 208,
250, 270, 272). A similar process governs the RyR chan-
nels in some invertebrate skeletal muscles (177, 260).
However, this is not the case in most vertebrate skeletal
muscle, where extracellular Ca 2
is not required for E-Ccoupling (9, 38, 91, 129, 177, 260). The primary role of the
DHPR in vertebrate skeletal muscles is acting as a voltage
sensor that directly (perhaps physically) modulates the
activation gate of nearby RyR1 channels.
A. DHPR-RyR Interaction
In skeletal muscle (Fig. 1), the DHPR and RyR1 are
thought to communicate via some sort of physical pro-
tein-protein linkage (39, 246, 247, 251, 253, 265). The
membranes of the t tubule and SR are juxtaposed and
separated by a small 10-nm gap. The cytosolic domain of the RyR1 channel spans this narrow gap (28, 29, 94 –97).
Electron microscopy (EM) studies show that the skeletal
DHPR in the t tubules are arranged in clusters of four
(tetrads). These tetrads are organized into distinct arrays.
The RyR1 channels in the SR membrane are arranged in a
corresponding fashion (28, 29, 94 –97, 247). The arrays of
DHPR and RyR align in certain fast-twitch skeletal muscle
such that every other RyR1 channel is associated with a
DHPR tetrad (28, 29, 247).
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It is thought that the skeletal DHPR physically con-
tacts the large cytoplasmic domain of the juxtaposed
RyR1 channel to form a linkage through which t-tubule
voltage-sensing information is transduced (246, 251–255,
265–267). A distinctive feature of this transduction mech-
anism is its speed. Signal transmission between the skel-
etal DHPR and RyR1 channel must occur during the very
brief (2 ms) skeletal muscle action potential. It may be
FIG. 1. Environment of the ryanodinereceptor channel in skeletal muscle. Thet tubule contains tetrads of dihydropyri-dine receptors (DHPRs). Every other RyR1 channel is associated with a DHPRtetrad. A t-tubule membrane voltagechange (V
M) activates the DHPR. Cal-
cium entry thorough the DHPR is not
important to RyR1 channel activation inskeletal muscle. Instead, the t-tubule V Msignal is thought to be transmitted to theRyR1 channel by a direct physical DHPR-RyR1 linkage. A specific cytosolic loop of the DHPR is thought to be involved inthis physical communication. The t-tu-bule V
M signal triggers the RyR1 chan-
nel to open and release the calciumstored inside the sarcoplasmic reticulum(SR). Calsequestrin (CSQ), a low-af finity
high-capacity calcium buffer, is found in-side the SR lumen near the RyR1 chan-
nels. The SR Ca
2
-ATPase (SERCA) usesthe energy stored in the ATP to pumpcalcium back into the SR.
FIG. 2. Environment of the RyR2channel in cardiac muscle. The t tubulecontains DHPRs and calcium extrusionmechanisms like the Na /Ca 2 ex-changer (Na-CaX). A t-tubule membrane
voltage change activates the DHPR, andthe resulting calcium entry triggers un-
derlying RyR2 channels to open. Theopen RyR2 channel releases the calcium,
stored inside the SR, into the cytoplasm.CSQ, a low-af finity high-capacity calciumbuffer, buffers calcium near the RyR2channels inside the SR. The SERCA usesthe energy stored in the ATP to pumpcalcium back into the SR. The Na-CaXuses the energy stored in the sodium gra-dient to move calcium out of the cell.
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this “need for speed” that has converted the physiological
role of the DHPR from Ca 2 channel to voltage sensor in
mammalian skeletal muscle.
In cardiac muscle (Fig. 2), there is about 1 DHPR for
every 5–10 RyR2 channels (29, 247, 313), and the DHPR
and RyR2 channels are not aligned in such a highly or-
dered fashion (29, 95, 247, 313). During the long cardiacaction potential (100 ms), the DHPR Ca 2 channel has
ample time to open and mediate a substantial Ca 2 influx.
This Ca 2 influx is ultimately the signal that activates the
underlying RyR2 channels via the CICR process (18, 40,
272). The involvement of a diffusible second messenger
(i.e., Ca 2) makes the DHPR-RyR2 signaling considerably
slower than the DHPR-RYR1 signaling. This lack of speed,
however, may provide a greater opportunity for regulating
DHPR-RyR2 signaling. In fact, pharmacological regulation
of DHPR-RyR2 signaling is fundamental to normal cardiac
function (18).
The expression of mutant DHPRs in mouse skeletalmuscle myotubes that lack the endogenous DHPR has
provided insights into which regions of the DHPR are
involved in DHPR-RyR1 interaction (1, 2, 106, 321). A
region within the cytosolic II-III loop of the DHPR’s -sub-
unit (residues 720 –765) appears to be critical. Further-
more, studies with myotubes lacking endogenous RyR1
channels showed that RyR2 or RyR3 could not substitute
for RyR1 in the DHPR-RyR interaction (216, 320, 356). The
expression of chimeric RyR channels (RyR1/RyR2) indi-
cates that regions R9 (2659 –3720) and R10 (1635–2636) of
RyR1 contain sequences involved in the DHPR-RyR1 in-
teraction (217, 246). Thus specific regions of the DHPRand RyR1 channels have been implicated in the DHPR-
RyR1 interaction. How these regions mediate the required
signal transduction remains to be determined.
The DHPR is not the only regulator of RyR1 channel
function. Like the RyR2 channel, the RyR1 channel (in the
absence of the DHPR) can be activated by cytosolic Ca 2
signals. This is important because more than half of the
RyR1 channels are not coupled to DHPRs (7, 183, 197,
200). These “uncoupled” RyR1 channels are thought to be
available to participate in the CICR process. It is generally
believed that CICR acts to amplify the DICR signal (i.e.,
that generated by the DHPR-RyR1 interaction). Interest-ingly, the presence of “coupled” and uncoupled RyR1
channels implies that RyR1 function is inherently hetero-
geneous “in vivo.” The amount of uncoupled RyR1 chan-
nels is not the same in all skeletal muscles. Slow-twitch
skeletal muscles may have three or more uncoupled RyR1
channels for each DHPR-linked RyR1 channel (7, 183, 197,
200). This may mean that these muscles have a greater
CICR component. This may be related to the substantially
slower rate of E-C coupling in slow-twitch muscle (104,
223, 227, 260, 370).
V. STUDY OF INTRACELLULAR
CALCIUM RELEASE
The first methods used to study Ca 2 transport
across cell membranes monitored radioisotope (45Ca) in-
flux/ef flux between two defined compartments (e.g., bath
and cytosol; Ref. 141). From these initial studies, it be-came clear that Ca 2 was sequestered into intracellular
organelles and that the cytosol represented an intermedi-
ary compartment with variable Ca 2 buffer properties.
The next generation of studies examined Ca 2 influx/
ef flux across the SR of mechanically or chemically
skinned muscle fibers. Here, the barrier of the plasma
membrane was eliminated and the cytosolic solution
could be controlled. The importance of ATP-mediated
Ca 2 uptake by the SR and some of the attributes of CICR
and even DICR were defined (80, 81, 150, 153, 151). Al-
most concurrently, methods for generating subcellular
fractionation of functional SR vesicles were developing.
Studies of Ca 2 loading and release from suspensions of
SR vesicles provided improved temporal resolution and
further revealed the intricacies of the SR Ca 2 release
process (121, 127, 138, 196, 198, 199, 234–236).
It was noticed by several investigators that the bind-
ing of the ryanodine alkaloid appeared to depend on the
activity level of the SR Ca 2 release channel (e.g., Refs.
53, 122). Experimental conditions that promoted SR Ca 2
release increased ryanodine binding. Experimental condi-
tions that inhibited SR Ca 2 release decreased ryanodine
binding. Subsequently, “nonequilibrium” ryanodine bind-
ing became a commonly used tool to assess RyR channel
activity (discussed in detail in Ref. 225). Thus a number of methods have been developed to study the SR Ca 2 re-
lease phenomenon in populations of RyR channels.
The development of the patch-clamp technique rev-
olutionized the study of ion channels, including the study
of the RyR channel. In this classic method, a small glass
pipette is placed on the surface of a cell to physically
isolate a small patch of membrane in which the function
of an individual ion channel can be measured. This method
has been widely applied to define the function of surface
membrane ion channels. The patch-clamp technique,
however, cannot be easily applied to study ion channels in
intracellular organelles. Instead, microsomes isolated
from cellular homogenates are fused into artificial planar lipid bilayers (206). The same instrumentation used to
record across the membrane patch is then used to record
across the artificial bilayer. Thus studies of single RyR
channel behavior became possible (detailed in sect. VI).
The recent development of laser scanning confocal
microscopy opened yet another chapter in the study of
intracellular Ca 2 release. Confocal imaging has made it
possible to visualize very localized intracellular Ca 2 re-
lease events (Ca 2 sparks; Refs. 50, 336). These local
events may arise from the opening of an individual chan-
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nel or more likely the concerted opening of a small num-
ber of RyR channels.
VI. STUDY OF SINGLE RYANODINE
RECEPTOR CHANNELS
Defining the characteristics of single RyR channels is
fundamental to understand the origin of many intracellu-
lar Ca 2 signaling phenomena. The amplitude of single
RyR channel current reveals the number of Ca 2 passing
through the channel per second. The open probability
( P o) of single RyR channels allows us to predict how
certain agonists and antagonists regulate its activity.
Dwell-time analysis of single RyR channel activity reveals
the durations of the open and closed events. Kinetic stud-
ies of single RyR channel activity reveal how fast it can
respond to a ligand stimulus (i.e., time constants and first
latencies) as well as its specific patterns of opening (i.e.,
modal gating or bursting behavior). Additionally, single
RyR channel studies can reveal the extent of homogeneity
or heterogeneity of individual channel function in the
overall RyR channel population (56, 60, 178, 238). These
types of information are generally not available when
Ca 2 signaling phenomena are studied in a population of
RyR channels. Thus single RyR channel studies provide
detailed information about the origin of intracellular Ca 2
signaling phenomena that simply cannot be obtained by
other means.
The study of single RyR channel behavior requires
the incorporation of native SR vesicles (enriched in RyR
channel) or purified RyR channel proteins into an artifi-cial planar lipid bilayer. The artificial planar bilayer is
generally formed across a small aperture that separates
two solutions. A patch-clamp amplifier is used to monitor
electrical signals that arise from the bilayer. Single RyR
channel activity is evident as a sudden appearance of
single-channel activity in the bilayer. The sudden appear-
ance of channel activity marks the moment a channel
protein has been incorporated into the bilayer. This meth-
odology is now widely used and has been described in
detail elsewhere (206).
The environment in which the single RyR channel
operates in the cell is complex (163). The resting cytosolic
free Ca 2 concentration is near 100 nM, and the free Ca 2
concentration inside the SR is thought to be near 1 mM
(196, 274). The cytosol contains a complex mixture of
salts (140 mM K , 1 mM Mg2, 5–10 mM ATP, and
various other nucleotides; Refs. 234, 371). The cell also
contains several proteins that are known to interact with
the RyR channel (e.g., DHPR, calmodulin, FK binding
proteins, triadin, kinases-phosphatases, calsequestrin, an-
nexin, etc.). Reconstructing or mimicking, at least in part,
this complex situation in vitro (i.e., during a planar bilayer
experiment) still needs to be done. Nearly all the single
RyR channel studies have been done under very simple
experimental conditions that do not reflect the channel’s physiological reality (e.g., Refs. 54, 56, 116, 145, 147, 291).
This fact should always be taken into consideration when
interpreting single RyR channel data.
How well does single RyR channel function in thebilayer reproduce the single RyR channel behavior in the
cell? The correlation between single RyR channel func-
tion in bilayers and SR Ca 2 release phenomena observed
in cells is surprisingly strong. For example, the primary
regulatory features of SR Ca 2 release in cells (e.g., its
Ca 2, Mg2, and ATP sensitivity) are clearly evident at the
single RyR channel level in bilayers. The actions of many
secondary pharmacological regulatory agents like caf-
feine, ruthenium red, and ryanodine in the cell are mim-
icked at the single RyR channel level in bilayers. The fast
gating and high conductance of single RyR channels in
bilayers is consistent with the speed and large amplitude
of intracellular Ca 2 release phenomena in cells (173).
There is growing evidence that many regulatory factors
and closely bound regulatory proteins are actually re-
tained when single RyR channels are isolated in native
membranes (e.g., Refs. 30, 189, 287, 300, 346, 348, 365).
Thus defining single RyR channel behavior in bilayers is
likely to provide insights into the origins of RyR-mediated
Ca 2 signaling in cells.
The RyR channel is usually isolated by differential
centrifugation of muscle homogenates. The RyR channel
is found in heavy microsomal fractions. The RyR-contain-
ing vesicles (microsomes) can then be fused into planar
lipid bilayers (36, 54, 56, 258, 291, 298). Alternatively, theRyR-containing vesicles can be detergent-solubilized
(CHAPS plus phospholipids mixtures) and the resulting
“ purified” RyR channels can then be directly fused in a
lipid bilayer (124, 135, 238) or reconstituted into lipo-
somes (145, 147). These proteoliposomes can then fuse
into planar lipid bilayers. Single RyR channel function has
been examined using all these approaches. An advantage
of using the SR vesicle approach is that the RyR channel
is present in its native membrane, increasing the likeli-
hood that closely associated regulatory proteins/factors
may still be present (e.g., Refs. 189, 327). A disadvantage
of using the SR vesicle approach is that the vesicles may
contain other types of ion channels, specifically K and/or Cl channels. The presence of other channels compli-
cates things considerably. Potential interference from
other channels is minimal if Ca 2 (or Ba 2) is used as the
charge carrier (with no other permeant ions present).
Many single RyR channel studies, however, chose to use
a monovalent charge carrier to boost signal-to-noise char-
acteristics. In this case, carefully selected ionic substitu-
tions are usually made to eliminate potential contamina-
tion from other channel types.
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In our experience, fusion of SR vesicles into planar
bilayers results in more stable/consistent single RyR
channel behavior compared with that obtained using
CHAPS-solubilized RyR channel preparations. As men-
tioned above, the use of SR vesicles requires that certain
specific steps be taken to avoid contaminating currents
from other ion channels. The first unitary Ca 2
currentrecordings associated with the RyR channel were made
after heavy SR vesicles were fused in a bilayer in the
presence of 50 mM Ca 2 and little else (291). The two
most common single RyR channel recording conditions
are listed below. One consists of a Ca-HEPES solution (50
mM Ca 2) on the luminal side with an isosmotic Tris-
HEPES solution on the cytosolic side of the channel. The
second consists of a Cs-methanesulfonate solution on
both sides of the channel. In both cases, contaminating
anion currents are avoided by the use of impermeable or
poorly permeable anions. In the first case, contaminating
cation currents are avoided by the presence of a nonper-
meable organic monovalent cation (Tris). In the second
case, contaminating K currents are avoided by using Cs
as a permeant monovalent cation. The use of Cs works
because SR K channels are either poorly permeable to
Cs or blocked by this ion, while RyR channels are highly
permeable to Cs (as well as other monovalent cations).
VII. PERMEATION PROPERTIES
The ability of the RyR channel to mobilize intracel-
lular Ca 2 depends on both its permeation and gating
properties. Its permeation properties include its unitaryconductance and ion selectivity. Its gating properties in-
clude its P o and the duration of individual open/closed
events. A single RyR channel with optimal permeation
properties would be ineffective at mobilizing Ca 2 if it
never opened (gated). Conversely, a single RyR channel
with optimal gating behavior would be worthless if it did
not allow Ca 2 to pass ef ficiently. The opening and clos-
ing of the RyR channel presumably involves the physical
motion of the protein. Ions are able to traverse its pore
only when the channel is in the open configuration. It is
generally assumed that movement of the gate does not
alter the nature of the pore (i.e., its diameter, length,
charge, etc.). It is in the pore where the channel discrim-ination between different ions occurs. The pore also con-
tains determinants that define how quickly particular ions
will pass. Specific RyR channel permeation properties are
described below. Specific features of single RyR channel
gating are discussed in later sections.
The permeation properties of the different RyR chan-
nel isoforms are quite similar. The RyR channels are
permeable to many different divalent and monovalent
cations (e.g., Refs. 167, 294, 330). In symmetrical solutions
containing a monovalent cation (e.g., 200 mM K , Na ,
or Cs) as the main permeant species, the unitary RyR
channel current-voltage relationship is linear with a slope
conductance usually 500 pS (167, 294). In asymmetric
solutions containing a divalent cation (e.g., 50 mM Ca 2,
Ba 2, or Sr 2) as the main permeant species, unitary RyR
channel current-voltage relationships have a slope con-ductance of 100 pS (330). These are very large conduc-
tance values. For comparison, high-conductance ion
channels of the plasma membrane such as the Ca 2-
activated K channels (i.e., maxi-K Ca channels) have a
maximal conductance of near 250 pS (157). The unitary
Ca 2 conductance of the L-type Ca 2 channel (i.e., DHPR)
in the surface membrane is no more than 20 pS (119).
Thus the RyR channels are very-large-conductance Ca 2
channels. This feature is probably fundamental to its
physiological role in cells, passing a large number of Ca 2
quickly.
The relative permeability of the RyR channels to
different monovalent and divalent cations has been de-
fined under bi-ionic conditions (e.g., Refs. 167, 294, 330).
The RyR channels show very little selectivity between
different monovalent cations. The RyR channels also
show very little selectivity between different divalent cat-
ions. However, the RyR channels are selective, albeit
poorly, between mono- and divalent cations ( P Ca / P K 6;
more permeable to Ca 2 than K ). This modest selectivity
for divalents is also unusual for a Ca 2 channel. The
typical surface membrane Ca 2 channel (e.g., DHPR) dis-
plays a much higher degree of discrimination between
monovalent and divalent cations ( P Ca / P K 20). The point
is that the RyR channel is not a highly selective Ca 2
channel. Intuitively, the apparent deficiency in Ca 2 se-
lectivity may be related to its high conductance. A high-
conductance channel (i.e., ions passing rapidly) would
have little time per ion to perform the steps needed to
discriminate between ions.
The relatively poor Ca 2 selectivity of the RyR chan-
nels does have important physiological ramifications. In
cells, Ca 2 is not the only ion that can permeate through
the open RyR channel pore. Other permeable cations
(Mg2, Na , and K ) are also present at substantial levels
(i.e., 1, 10, and 140 mM, respectively). Thus Ca 2 com-
petes with Mg2, Na , and K for occupancy of the RyR
channel pore. The consequence is that the unitary Ca 2
current carried by a single RyR channel in the cell (i.e., in
the presence of multiple permeant ions) will be consider-
ably less than measured under the traditional simple re-
cording conditions used in bilayer studies (e.g., Refs. 256,
259, 295, 330).
Tinker et al. (329) were the first to examine how
unitary Ca 2 current through the RyR2 channel is affected
by the presence of other permeant ions. They estimated
that unitary Ca 2 current (at 0 mV) under physiological
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conditions would be 1.4 pA. This still quite large current
was explained using a Eyring-rate model (328) assuming
that some mechanism allows the association rate of Ca 2
at the mouth of the pore to exceed, by about an order of
magnitude, the normal diffusional limit. A more recent
examination of unitary Ca 2 current through the single
RyR2 channel suggests that unitary Ca 2
current (at 0mV) under physiological conditions may be closer to 0.5
pA (201). This is important because accurately defining
the amplitude of the unitary Ca 2 current through a single
RyR channel is fundamental to understanding the origin
of local intracellular Ca 2 signaling events (e.g., Ca 2
sparks) in cells. The unitary RyR Ca 2 current value is
central to the debate whether Ca 2 sparks arise from the
opening of one RyR channel or the concerted opening of
many RyR channels.
The larger unitary RyR channel Ca 2 current predic-
tion suggests that a mechanism to enhance permeation
may possibly exist (328). The smaller unitary Ca 2 cur-
rent prediction indicates that perhaps there is no need for
any permeation enhancement mechanism (201). What
might the permeation enhancement mechanism be? Sev-
eral possible permeability enhancement mechanisms
have been proposed. It has been argued that the RyR2
channel may contain a large charged vestibule that dielec-
trically focuses ions near the pore’s mouth (328). Alter-
natively, dielectric focusing by fixed surface charges
could occur near the RyR channel’s mouth in the absence
of a large vestibule structure (337). It has also been sug-
gested that a single RyR channel contains multiple paral-
lel conduction pathways (i.e., a multibarrel pore). The
later possibility was supported by the initial 3-D RyRchannel reconstructions that suggested the presence of
multiple conduction pathways (342). Furthermore, On-
drias et al. (228) suggested that the four commonly ob-
served subconductance states of the RyR channel might
correspond to uncoordinated opening of pores in the
individual RyR monomers of the tetrameric channel com-
plex. They suggested that the presence of the FK-binding
protein synchronizes the openings of these separate per-
meation pathways. However, recent and more detailed
3-D reconstruction data suggest that the RyR channel
structure contains a single central pore (i.e., the RyR is
not a multibarrel channel; Refs. 230, 270, 275). However,
the possibility that the channel contains a large vestibuleand/or surface charges that impact ion permeation still
exists and should not be discounted.
VIII. ACTION OF RYANODINE
The alkaloid ryanodine binds with high af finity to the
RyR proteins (225). Its binding induces a complex change
in single RyR channel function. This change is similar in
all three RyR channel isoforms. Ryanodine binding in-
duces very-long-duration open events and simultaneously
reduces ion conductance through the pore (e.g., Refs. 259,
294). This dual impact on single-channel function (i.e.,
slower gating, reduced conductance) suggests a compli-
cated mode of action. This is also evidenced by the rather
complicated ryanodine dose dependency. Low doses of
ryanodine (10 nM) are reported to increase the fre-quency of single RyR channel openings to the normal
conductance level (34). Intermediate ryanodine doses
(1 M) are reported to induce the classical ryanodine
action described above (34, 259, 294). High doses of ry-
anodine (100 M) are reported to lock the channel in a
closed configuration (368).
The fact that ryanodine binding alters single-channel
conductance suggests that ryanodine binds in or near the
RyR channel pore. In fact, the ryanodine-binding site has
been experimentally localized to the COOH terminus of
the protein (37), the region of the RyR protein thought to
contain the structural determinants of the pore. Altered
conductance suggests that ryanodine binding somehow
changes the nature of the permeation pathway. This idea
has also been experimentally confirmed. Two indepen-
dent studies have indicated that ryanodine binding actu-
ally changes the effective diameter of the RyR channel
pore (332, 337).
The complex action of ryanodine may be related to
how the alkaloid binds to the channel. It is generally
assumed that each RyR monomer contains one high-af fin-
ity ryanodine binding site (K D 50 nM; Refs. 147, 225). It
is possible that cooperative interaction between these
different ryanodine binding sites is what generates the
observed complexity (53, 242). Some ryanodine bindingstudies suggest the existence of additional lower (K D 1
M) af finity ryanodine binding sites (53, 147, 225, 242).
Thus the RyR channel complex may contain multiple
ryanodine binding sites. One interpretation suggests that
ryanodine binding to the high-af finity site depends on
whether or not the RyR channel is in the open con figura-
tion (53). It may be that conformational changes related
to the opening of the RyR channel exposes (or improves
access to) the high-af finity ryanodine binding site. This
would impart a use dependence to the kinetics of ryano-
dine binding, allowing “nonequilibrium” ryanodine bind-
ing to be used as a monitor of RyR channel function (as
described previously).
IX. OVERVIEW OF RYANODINE
RECEPTOR REGULATION
The RyR channels are modulated by numerous fac-
tors, including a number of physiological agents (e.g.,
Ca 2, ATP, and Mg2), various cellular processes (e.g.,
phosphorylation, oxidation, etc.), and several pharmaco-
logical agents (e.g., ryanodine, caffeine, and ruthenium
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red). Some of these modulatory factors are discussed
below.
A. Cytosolic Ca2
The action of Ca
2
on the RyR channel is complex.The Ca 2 turns on, turns off, and conducts through this
channel. Steady-state single RyR channel activity is gen-
erally a bell-shaped function of cytosolic Ca 2 concentra-
tion (35, 46, 54, 56, 60, 135, 161, 224, 238, 285, 294, 333).
The RyR channels are activated by low Ca 2 concentra-
tions (1–10 M) and inhibited by high Ca 2 concentra-
tions (1–10 mM). It has been reported that individual
RyR1 channels do not all respond in the same way (i.e.,
they are functionally heterogeneous) to activating cytoso-
lic Ca 2 levels (56, 161, 178, 238). In contrast, individual
RyR2 and RyR3 channels respond much more homoge-
neously to cytosolic Ca 2 (46, 54, 56, 135, 161). Inhibition
at high cytosolic Ca 2
concentrations of the RyR1 is alsodifferent from that of RyR2 and RyR3 channels. The RyR1
channel is almost entirely inhibited by 1 mM Ca 2,
whereas the RyR2 and RyR3 channels are not (35, 46, 54,
56, 60, 135, 161, 224, 225, 238, 264). Substantially higher
Ca 2 levels are required to inhibit the RyR2 and RyR3
channels (46, 54, 56, 135, 161). The reported half-maximal
inhibiting concentrations for the RyR2 and RyR3 channels
range from 2 to 10 mM. It is not clear that such high
cytoplasmic Ca 2 concentrations are ever reached in the
cells. Thus the physiological role of this very high Ca 2
inhibition is not yet clear. Nevertheless, high Ca 2 inhibi-
tion of the RyR2 channels remains a controversial topic.
This is further discussed in section XV .
B. Luminal Ca2
Early Ca 2 release studies suggested that Ca 2 bind-
ing to the luminal surface of the RyR channel may regu-
late the channel (64, 65). Single RyR channel studies have
confirmed the existence of luminal Ca 2 regulation (51,
110, 286, 285, 333, 341). It appears that the sensitivity of
RyR channel to certain cytosolic agonists increases at
high luminal Ca 2 levels (110, 286). Trypsin digestion of
the luminal side of the RyR channel suggested the pres-ence of both activating and inactivating divalent sites
(51). It is possible that the luminal Ca 2 effects are me-
diated, at least in part, by associated proteins like calse-
questrin (14, 314) or junctin (364).
It was also reported that luminal Ca 2 moving
through the channel feeds back and interacts with cyto-
solic Ca 2 binding sites (333). Thus luminal Ca 2 levels
may affect single RyR channel function by interacting
with luminal binding sites, with associated luminal pro-
teins, or with cytosolic binding sites (i.e., feed-through
regulation). Distinguishing between these possibilities is
the challenge.
C. ATP and Mg2
Cytosolic ATP is an effective RyR channel activator (57, 60, 291, 297, 298, 353). Cytosolic Mg2 is a potent RyR
channel inhibitor (57, 60, 158, 291, 353). The cytosol of
most cells contains 5 mM total ATP and 1 mM free
Mg2. This means that most ATP is in its Mg2-bound
form. Free ATP (300 M in the cytosol) is the species
that binds to and activates the RyR channel (297, 298).
The action of ATP and Mg2 on single RyR channel func-
tion is isoform specific. Free ATP is a much more effec-
tive activator of the RyR1 channel than the RyR2 channel
(57, 60, 291, 297, 353). The RyR3 channel is also less ATP
sensitive than the RyR1 channel (46, 135, 297). The Mg2
action on single RyR channels is complicated (57, 158).First, Mg2may compete with Ca 2 at the Ca 2 activation
site, shifting the Ca 2 sensitivity of the channel (57, 60,
158, 353). Second, Mg2 competes with Ca 2 at the high
Ca 2 inhibition site (57, 158). The high Ca 2 inhibition site
does not discriminate between different divalent cations.
The RyR1 channel is substantially more sensitive to this
action of Mg2 than the RyR2 or RyR3 channels (57, 135).
In the presence of physiological levels of Mg2 and ATP,
the RyR1 channel requires less Ca 2 to activate than the
RyR2 or RyR3 channel (57, 257, 320, 353).
D. Redox Status
Various redox processes may also modulate single
RyR channels. The RyR monomers contain 80 –100 cys-
teine residues (315a, 318). Many of these are suitable for
modification by oxidants. There is evidence that oxidant
and reducing agents do indeed impact single RyR channel
function (182, 222, 349), including the channel’s Mg2
sensitivity. There is also evidence that oxidants may pre-
vent certain closely associated regulatory molecules, like
calmodulin, to bind to the RyR channel (363). Recently, it
has been suggested that nitric oxide (NO) may modulate
the redox regulation of the RyR channels (79, 118, 352). It
was proposed that in vivo initial NO release may activate
RyR channels and subsequent increases in NO levels in-
hibit RyR channel activity (118). Thus there is a growing
interest in identifying the cysteine residues that may be
involved in RyR channel oxidation and/or S -nitrosylation
(352). It is quite possible that redox modulation of single
RyR channels represents an important regulatory mecha-
nism. It is clear that additional information is needed in
this area.
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E. Cyclic ADP-ribose
Cyclic ADP-ribose (cADPR) appears to play an im-
portant role in many nonmuscle systems (164). The
cADPR molecule, a metabolite of NADP, has been found
to act as a powerful intracellular Ca 2 release agent in sea
urchin eggs, and its action is thought to be mediated byRyR or RyR-like channels (164). The direct action of
cADPR on RyR channels from muscle systems is contro-
versial. An early report indicated that cADPR dramatically
activates single mammalian RyR channels (203). How-
ever, subsequent studies reported only minor effects of
cADPR (284) or no impact of cADPR at all (58, 100, 209).
The controversial reports may suggest a requirement of
an unrecognized cofactor and/or a potential interaction
with some closely associated regulatory factor (i.e., cal-
modulin, FK-506 binding protein; Refs. 58, 164). When
studied in intact cellular systems (that contain endoge-
nous calmodulin and FK-binding proteins), cADPR had
only indirect action on RyR channel function (107, 126,175). It appeared that cADPR activated the Ca 2-ATPase
(175), which indirectly activated the RyR channel by in-
creasing luminal SR Ca 2 levels.
F. Phosphorylation/dephosphorylation
Analysis of the RyR protein sequence reveals that it
contains many consensus phosphorylation sites (315a,
318). For more than a decade, the effects of exogenously
applied kinases and phosphatases on single RyR channel
behavior have been examined (60, 114, 170, 189, 298, 315,
339, 348). The capacity of protein kinase A (PKA) toactivate the RyR1 channel has been reported (60, 298).
This is consistent with the observation that PKA signifi-
cantly increases depolarization-induced Ca 2 release
from skeletal muscle SR vesicles (125). However, studies
in skinned and intact skeletal muscle fibers have not
supported this idea (27). The capacity of calmodulin
kinase and PKA to activate the RyR2 channel has also
been studied. Contradictory results have been reported
for calmodulin kinase (70, 114, 170). Similarly, some re-
ports on PKA action find activation of RyR2 channels
(114), whereas others report a slight decrease in the
steady-state P o of RyR2 channels (339). Still others sug-
gest PKA destabilizes RyR2 channel openings (i.e., pro-mote substating) via phosphorylation-induced dissocia-
tion of FKBP12.6 (189). Yet another report shows that
PKA phosphorylation does not impact the RyR-mediated
Ca 2 spark in intact cardiac myocytes (166). The reason
for the appearance of such contradictory and confusing
results is unclear. It is possible that different experimen-
tal conditions are responsible. It is safe to conclude that
the impact of phosphorylation and/or dephosphorylation
on single RyR channel behavior will be debated for some
time.
X. REGULATION BY CLOSELY
ASSOCIATED PROTEINS
A number of different proteins may be associated
with and modulate the RyR channels (60, 63, 136, 142, 144,
169, 181, 183, 185, 196, 231, 257, 287, 334, 343). However,
only a few of these proteins have been thoroughly studiedat the single-channel level. These few “well-studied” pro-
teins include calmodulin, calsequestrin, FK-506 binding
protein, and certain fragments of the DHPR. A brief sur-
vey of how these proteins may interact with the RyR
channel is provided below.
A. Calmodulin
Calmodulin (CaM) was the first peptide found to
interact with single RyR channels in lipid bilayers (295).
CaM appears to bind the RyR in stoichiometric propor-
tions (11, 207, 334). It binds to sites in the RyR channelcytoplasmic assembly that are 10 nm from the putative
entrance to the transmembrane pore (343). Application of
CaM can either activate (at low Ca 2 levels) or inhibit (at
high Ca 2 levels) the RyR1 and RyR3 channels (46, 99,
295, 334). For the RyR2 channel, only inhibitory effects of
CaM have been reported (99, 295). It was reported that
oxidation modifies RyR1 channel behavior and perhaps
its interaction with CaM (117, 363). It has also been
suggested that CaM may somehow protect the RyR1 chan-
nel from oxidative modifications during periods of oxida-
tive stress (363). CaM also appears to bind to the DHPR
and consequently has also been implicated in the DHPR-
RyR1 interaction (117, 269). A potential role of CaM inmodulation of the RyR2 channel during E-C coupling is
not yet clear.
B. Calsequestrin
Calsequestrin is the main Ca 2 binding protein inside
the SR. Calsequestrin has a large number of acidic amino
acids that permit it to coordinately bind 40–50 Ca 2 /
molecule (181, 358). Calsequestrin is also known to ag-
gregate in the presence of divalent cations (190). Electron
microscopy studies suggest that calsequestrin aggregates
are present in the regions of the SR (i.e., terminal cister-nae) known to contain RyR channels (96). It has been
suggested that Ca 2- and pH-dependent conformational
changes in calsequestrin may modulate RyR channel ac-
tivity (61, 120). It also has been suggested that calseques-
trin action on the RyR channel may require the presence
of triadin, another RyR-associated protein (365). Thus
there is disagreement concerning the nature of the calse-
questrin-RyR interaction.
Some studies in lipid bilayers suggest that calseques-
trin activates the RyR channel (139, 226, 314). However,
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described previously, the DHPR-RyR interaction is clearly
involved in the signaling that links surface membrane
excitation to SR Ca 2 release in striated muscle. The
nature of the DHPR-RyR interaction is tissue specific. In
skeletal muscle, the interaction is thought to involve a
direct physical link between the two proteins. In cardiac
muscle, the DHPR Ca 2
channel mediates a small Ca 2
influx that activates the RyR2 channel. Our attention here
will be focused on the physical DHPR-RyR1 link in skel-
etal muscle.
Tanabe et al. (321) showed that the cytoplasmic II-III
loop of the skeletal DHPR 1-subunit is required for the
functional DHPR-RyR1 interaction. Although other re-
gions of the DHPR may be involved (62, 165, 289), the
potential role of the II-III loop has been studied most to
date. Peptide fragments that correspond to the II-III loop
activate single RyR1 channels in planar lipid bilayers (171,
172). Interestingly, different regions of the peptide appear
to have different actions on RyR1 channel function. One
region called peptide A activates single RyR1 channel (71,
171, 172, 262). Another region called peptide C blocks the
activating action of peptide A (71, 171, 262). Additional
insights have been gained from examining the action of
the scorpion peptide toxin imperatoxin A, whose struc-
ture has similarities to that of peptide A (108). Like pep-
tide A, imperatoxin A activates single RyR1 channel in
planar bilayers (108). The toxin binds with high af finity to
the RyR1 channel but at a site that appears to be quite
distant from the RyR1 regions thought to interact with the
DHPR (263). If peptide A and imperatoxin A bind to the
same site, then the distant location of this site is dif ficult
to reconcile with our current understanding of the DHPR-RyR1 interaction. Recently, the interpretation of the phys-
iological role of peptide A on RyR1 channel function was
further complicated. Grabner et al. (106) showed that a
chimeric DHPR, lacking the peptide A region, supported
normal DHPR-RyR1 signaling when expressed in dysgenic
myotubes (i.e., myotubes lacking endogenous DHPR).
Thus the role of peptide A in the DHPR-RyR1 interaction
has yet to be clearly established.
XI. CALCIUM REGULATION PARADOX
Although the RyR channels are modulated by manyfactors, Ca 2 regulation plays a central role in all RyR-
mediated signaling cascades. In some cases (i.e., cardiac
muscle), the regulating Ca 2 signal initiates RyR channel
activity. In other cases (i.e., skeletal muscle), the regulat-
ing Ca 2 signal may amplify RyR channel activity. There is
also evidence that regulating Ca 2 signals may terminate
RyR channel activity. The importance of understanding
how Ca 2 regulates the RyR channels cannot be overstated.
Our discussion of RyR2 channel Ca 2 regulation
starts with the classical work of Fabiato (80, 81), which
defined the Ca 2 regulation of the SR Ca 2 release pro-
cess in a skinned cardiac muscle preparation. This work
showed that the amplitude and duration of trigger Ca 2
stimulus finely grades the CICR process (i.e., Ca 2-in-
duced Ca 2 release). Specifically, the amount of Ca 2
released from the SR was a bell-shaped function of trigger
Ca 2
stimulus amplitude, and that trigger Ca 2
stimulusspeed scaled this relationship.
Intuitively, the CICR process should be self-regener-
ating because the Ca 2 released by a RyR2 channel should
feedback and further activate the channel or its neigh-
bors. However, self-regeneration of CICR is not observed
in cells. This is the Ca 2 regulation paradox. The impli-
cation is that some sort of negative-feedback mecha-
nism(s) must exist to counter the inherent positive feed-
back of CICR. Fabiato (81) proposed that the negative-
feedback mechanism was Ca 2-dependent inactivation.
The idea was that two different Ca 2 binding sites (acti-
vating and inhibitory, respectively) regulate the Ca 2 re-
lease channel (i.e., RyR2). The Ca 2 activation process
was thought to have a fast action and a relatively low
af finity. The Ca 2 inactivation process had a slower ac-
tion but a relatively high Ca 2 af finity (81). Consequently,
a fast Ca 2 stimulus transiently activates the channel
because the channel would be “turned on” as Ca 2 occu-
pies the activation site and then “turned off ” as Ca 2more
slowly acts at the inactivation site. A slow Ca 2 stimulus
would not effectively activate the channel because inac-
tivation would “keep pace” with the activation process. At
very high Ca 2 levels (100 M), the inactivation sites
would be saturated, leaving the channel in an inactivated
state. The mechanism of Ca 2
-dependent inactivation asenvisioned by Fabiato (81) implies that the channel be-
comes refractory (unable to respond to further Ca 2 stim-
ulation). Recovery from the refractory state would require
removal of the Ca 2 stimulus and enough time for some
sort of recovery process (i.e., repriming) to occur. This
scheme is still widely discussed and is often applied to
explain the complex Ca 2 regulation of many RyR-medi-
ated Ca 2 signaling events.
Despite its popularity, the existence of high-af finity
Ca 2-dependent inactivation has been challenged. Studies
on patch-clamped cardiac myocytes have not conclu-
sively confirmed the existence of the Ca 2-dependent
inactivation phenomenon (208, 214, 360). For example,the capacity of a second Ca 2 stimulus to trigger SR Ca 2
release was shown to be independent of the interval
between the first and second stimuli (214). The argument
here is that if the SR Ca 2 release channels were inacti-
vated after the first stimulus, then the effectiveness of the
second stimulus should be affected. In another study
(360), a prolonged triggering Ca 2 signal was used to
inactivate the Ca 2 release process. A subsequent trigger
Ca 2 signal, however, reactivated the apparently inacti-
vated release process (i.e., the process was not refractory).
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Still, it is possible that the second Ca 2 stimulus (in both
cases above) was activating neighboring Ca 2 release
sites that did not participate in the first Ca 2 stimulus. In
any event, studies at the whole cell level have not con-
clusively confirmed or dismissed the existence of Ca 2-
dependent inactivation. Studies of RyR2 channel Ca 2
regulation at the single-channel level promised to resolvethis question.
XII. STEADY-STATE CALCIUM DEPENDENCE
As discussed above, several laboratories have de-
fined the Ca 2 dependence of single RyR1 and RyR2
channels under steady-state conditions (e.g., Refs. 54, 56,
161, 256). In the absence of other modulating agents,
these studies show that there is little spontaneous chan-
nel activity at low cytosolic free Ca 2 concentrations
(100 nM). This is consistent with the rarity of sponta-
neous Ca 2
release events (i.e., Ca 2
sparks) at restingintracellular Ca 2 levels in cells (25, 50). There is substan-
tial spontaneous activity of single RyR channels at steady-
state Ca 2 concentrations in the micromolar range. The
degree of spontaneous activity peaks at 10 M. The
apparent K D of single RyR channel Ca 2 activation is
typically between 0.5 and 5 M. This is consistent with the
Ca 2 sensitivity of SR Ca 2 release activation in skinned
cardiac cells (81) and isolated cardiac and skeletal SR
vesicle preparations (54, 199, 234–236, 355).
The activity of single RyR1 channel decreases to near
zero if the steady-state Ca 2 concentration is elevated to
1 mM (54, 56, 161, 238). This is not true for the RyR2 or
RyR3 channels. Substantial inhibition of these channels isonly observed when the steady-state Ca 2 concentration
exceed 5–10 mM (54, 56, 135, 161, 238). It is unlikely that
the free Ca 2 levels in the cytosol of a cell would ever
exceed the 1 mM mark because the Ca 2 sources used to
elevate cytosolic Ca 2 (i.e., extracellular solution and SR
lumen) contain only 1 mM Ca 2. Thus the physiological
relevance of such low-af finity Ca 2 inhibition is question-
able. It is unclear how such low af finity of Ca 2 inhibition
(as observed at the single-channel level) is related to the
apparent high-af finity Ca 2-dependent inactivation ob-
served in cells (81). However, the RyR channels operate in
a dynamic (not stationary) Ca
2
signaling environment.Thus steady-state measurements of channel activity (over
minutes) may not reveal all the intricacies of single RyR
channel Ca 2 regulation in cells (on a millisecond time
scale).
XIII. CALCIUM ACTIVATION KINETICS
Several groups have defined the kinetics of single
RyR2 channel Ca 2 regulation in bilayers (e.g., Refs. 112,
113, 159, 264, 283, 339). There is considerably less data
addressing the kinetics of single RyR1 channels (e.g., Ref.
340) and none concerning single RyR3 channel function.
Consequently, the discussion here focuses mainly on the
kinetics of RyR2 channel Ca 2 regulation.
A. Rate of Ca
2
Activation
Fast Ca 2 stimuli were applied to single RyR2 chan-
nels using two different methodologies. One methodology
generates fast Ca 2 stimuli by liberating Ca 2 from caged-
Ca 2 compounds using laser flash photolysis (112, 339,
340). The other methodology generates fast Ca 2 stimuli
by mechanically changing the solution near the RyR2
channel (159, 264, 283). The RyR2 channel activates with
a time constant of 1 ms when the Ca 2 is very rapidly
changed via flash photolysis of caged-Ca 2 and slightly
slower ( 2–20 ms) when Ca 2 is changed by a mechan-
ical solution change technique. These numbers are fairly
consistent and are generally in agreement with whole cell
studies that clearly show that Ca 2 activation of SR Ca 2
release occurs on a millisecond time scale.
Interestingly, the reported RyR2 channel Ca 2 acti-
vation time constants vary with the speed of the applied
Ca 2 stimulus. For example, Laver and Curtis (159) ap-
plied relatively slow mechanical Ca 2 changes and re-
ported a time constant of 20 ms. Sitsapesan et al. (283)
applied slightly faster mechanical Ca 2 changes and re-
ported a Ca 2 activation time constant of 10 ms.
Schiefer et al. (264) applied even faster mechanical Ca 2
changes and reported a time constant between 2 and 5
ms. The fastest Ca 2
changes, however, were appliedusing the flash photolysis method. These studies report a
Ca 2 activation time constant of 1 ms (112, 113, 339).
The interpretation of the mechanical solution change
data described above must consider the presence of un-
stirred layers immediately in front of the bilayer. In some
of the mechanical solution change studies (159, 283), the
size of the unstirred layers clearly impacted the rate of
Ca 2 change in the microenvironment of the channel.
Unfortunately, concerns about the impact of unstirred
layers have rarely been discussed. Unstirred layers would
tend to slow the measured Ca 2 activation time constant.
Unstirred layers, however, do not complicate the inter-
pretation of the flash photolysis data, but there is another concern here. The Ca 2 waveform applied by flash pho-
tolysis is not a simple square step. Instead, there is a fast
Ca 2 overshoot at its leading edge (77, 78, 194, 372). This
very brief Ca 2 overshoot (lasting 150 s) likely accel-
erates the initial opening transition of the RyR2 channel.
The overshoot in these studies would tend to speed up the
measured Ca 2 activation time constant. Thus there are
studies (flash photolysis) that may overestimate the Ca 2
activation kinetics as well as studies (mechanical solution
change) that may underestimate it.
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What is the real RyR2 channel Ca 2 activation kinet-
ics? The single RyR2 channel Ca 2 activation time con-
stant ( 2–5 ms) to a steplike Ca 2 stimulus is probably
best represented by the data of Schiefer et al. (264). This
study applied very fast mechanical Ca 2 changes on very
small-diameter bilayers alleviating unstirred layer con-
cerns. These types of experiments, however, have never been repeated. The physiological Ca 2 trigger in cells is
clearly not a simple square Ca 2 step. The RyR2 channels
in vivo are governed by the very fast transient Ca 2
changes that occur near DHPR (i.e., L-type) Ca 2 chan-
nels when they flicker open (302). It has been estimated
that the free Ca 2 concentration near the open mouth of
a DHPR Ca 2 channel can reach very high levels (100
M) when it is open and fall very rapidly (10 s) after
the channel closes. Interestingly, the fast Ca 2 overshoot
generated by flash photolysis has similar characteristics
(78). The rise time of the fast Ca 2 overshoot has a time
constant of 30 s, and the Ca 2 overshoot can theoret-
ically peak at 50–100 M before it decays with a time
constant of 150 s. Thus the fast Ca 2 overshoot may be
a reasonable reproduction of the free Ca 2 changes that
initiate RyR2 channel activity in the cell. Therefore, the
single RyR2 channel Ca 2 activation time constant to
physiological Ca 2 stimuli may be best represented by the
flash photolysis data ( 1 ms; Ref. 112).
B. Rate of Ca2 Deactivation
Fast Ca 2 elevations clearly turn on single RyR2
channels rapidly (112, 264). An equally important questionis, How quickly do single RyR2 channels turn off (deacti-
vate) in response to fast Ca 2 reductions? Intuitively, it
will take some time for Ca 2 to “fall off ” the Ca 2 activa -
tion site and for the channel to move into a closed con-
figuration. The Ca 2 deactivation kinetics of single RyR2
channels have been explored using both fast solution
exchanges (264, 283) and flash photolysis (340). Sitsape-
san et al. (283) reported that the time constant of RyR2
channel Ca 2 deactivation was 10 ms, about the mea-
sured rate of the Ca 2 change. Schiefer et al. (264) re-
ported that RyR2 channel Ca 2 deactivation occurred
with a time constant of 6 ms, 10 times slower than
measured Ca 2 change. Velez et al. (340) photolyzedDiazo-2 (a caged Ca 2 chelator) to reduce the local free
Ca 2 concentration near a single RyR2 channel in planar
bilayers. They reported a deactivation time constant of 5.3
ms, 10 times slower than measured Ca 2 change. Thus
there is reasonable consensus that single RyR2 channels
deactivate rapidly with a time constant of 5– 6 ms in
response to a fast Ca 2 reduction.
The point is that the Ca 2 activation and deactivation
kinetics of single RyR2 channels in vitro are suf ficient for
the channel to respond to the very brief (1 ms) trigger
Ca 2 concentration changes, the kind of local trigger
Ca 2 signals that are thought to govern RyR2 channel
activity in vivo (302).
XIV. FEEDBACK CALCIUM REGULATION
One suggestion is that the Ca 2 interacting with the
Ca 2 activation site is different from the Ca 2 that passes
through the open RyR channel pore. Another hypothesis
is that the Ca 2 activation site may “see” the Ca 2 flux
that passes through its own open pore. In other words, the
RyR channel may operate in a local “common Ca 2 pool”(302). There are several whole cell observations that are
consistent with this latter common Ca 2 pool idea (25,
168, 237, 344).
Is this type of self-feedback Ca 2 regulation seen in
single RyR channel studies? Most single RyR studies have
been done under conditions that generate superphysi-
ological unitary Ca 2
currents (285, 290 –292, 306). The pertinent single RyR channel studies were done with huge
Ca 2 driving forces that are more than 50 times greater
than those thought to exist in cells. This would increase
the possibility of feedback Ca 2 regulation in ways that
do not exist in the cell. This fact must be considered here.
The steady-state cytosolic Ca 2 sensitivity of the
RyR1 or RyR2 channels appears to be the same whether
the charge carrier is Ca 2 or a monovalent cation (58, 161,
290, 294, 306). This observation implies, but does not
prove, that charge carrier identity does not impact RyR
channel Ca 2 regulation. Sitsapesan and Williams (285)
have evaluated single RyR2 channel gating in the pres-
ence of different luminal Ca 2 concentrations. They con-cluded that P o and open/closed lifetimes of Ca 2-acti-
vated, Ca 2-conducting RyR1 channels were independent
of the Ca 2 flux through the channel. In contrast, Tripathy
and Meissner (333) reported that the duration of single
RyR1 channel openings was significantly longer in the
presence of even a relatively small Ca 2 flux through the
pore. They go on to predict that the RyR1 channel’s Ca 2
activation site must be 75 nm from the pore to explain
their results. Interestingly, the RyR channel complex is
only 25 nm square (230, 249, 342). This may indicate that
the RyR1 channel’s Ca 2 activation site may be in some
sort of protected pocket that shields the site from theCa 2 coming through the pore. In any event, it is clear that
additional data are required to establish whether or not
feedback Ca 2 regulation occurs.
XV. POTENTIAL NEGATIVE
CONTROL MECHANISMS
It is clear that some sort of RyR channel negative
control mechanism must exist to counter the inherent
positive feedback of the CICR process (81, 105, 214, 255,
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265, 302, 303, 307). There is a growing consensus that the
negative control that counters the inherent positive feed-
back of CICR is actually a composite of factors/processes
that act in concert to terminate local RyR-mediated Ca 2
release. This latter idea is consistent with the fact that no
individual negative control mechanism appears suf ficient
by itself to explain the termination of local Ca 2
release.Several candidate RyR channel negative control mecha-
nisms are discussed below.
A. Ca2-Dependent Inactivation
The first negative-feedback mechanism considered
was Ca 2-dependent inactivation (81). It was reported
that the SR Ca 2 release process is substantially inacti-
vated at steady-state Ca 2 concentrations as low as 60 nM
in skinned cardiac muscle fibers (81). However, single
RyR2 channel studies in bilayers have forwarded no evi-
dence of Ca 2
-dependent inactivation at such low Ca 2
concentrations (54, 161, 256). Also, subsequent studies on
intact cells presented contradictory results (109, 208, 214,
280). For example, Lukyanenko and Gyo ¨ rke et al. (173)
imaged permeabilized ventricular myocytes using a con-
focal microscope and showed that elevation of resting
Ca 2 levels resulted in an increase in RyR2-mediated Ca 2
spark activity, not the decrease expected if Ca 2-depen-
dent inactivation exists. Thus the existence of high-af fin-
ity Ca 2-dependent inactivation is debated. The existence
of low-af finity Ca 2-dependent inactivation is not. Single
RyR1 and RyR2 channel data clearly demonstrate the
existence of low-af finity Ca 2 inhibition under steady-
state experimental conditions. Single RyR1 channelsclearly turn off as the cytosolic free Ca 2 concentration is
elevated toward the 1 mM mark (196, 253, 307). This
low-af finity Ca 2 inhibition could come into play if local
Ca 2 levels in the cell reach the 0.6 –1 mM range (156).
For the RyR2 channel, the situation is different. Single
RyR2 channels turn off only after free Ca 2 concentra-
tions reach much higher levels (5–10 mM; Refs. 12, 54, 56,
161, 199, 256). These levels are probably never realized in
a cell. Thus Ca 2-dependent inactivation as a potential
negative control mechanism that may account for (or
contribute to) the termination of local RyR-mediated
Ca
2
release events is still not clearly established.
B. Stochastic Attrition
Stochastic attrition refers to the inherent random
closing of an individual ion channel. Stern (302) demon-
strated using mathematical modeling that a SR Ca 2 re-
lease unit composed of one or relatively small number of
RyR2 channels will turn off spontaneously as a result of
reduced local Ca 2 levels resulting from the stochastic
closures of individual RyR2 channels in the cluster. Thus
stochastic attrition of single RyR2 channel activity could
contribute to terminating a local Ca 2 release event. This
process, however, would be very sensitive to the number
of channels involved and the positive feedback gain of
CICR in the channel cluster. Stern (302) showed that
stochastic attrition was suf ficient to terminate local Ca 2
release only if there were 10 RyR2 channels per cluster.If release units are composed of 10 –30 or more channels
(31, 95, 174), then the likelihood of termination solely by
stochastic attrition is low. Experimentally, Lukyanenko et
al. (176) showed that the rate of Ca 2 release termination
is proportional to the magnitude of the release event (i.e.,
large events terminate faster than small events). Addition-
ally, Sham et al. (273) demonstrated that local Ca 2 re-
lease events terminate rapidly despite the presence of a
sustained trigger Ca 2 signal. These results are inconsis-
tent with stochastic attrition playing a major role in con-
trol of the CICR process. Nevertheless, stochastic attri-
tion may still contribute to local Ca 2 release termination
if it acts in combination with other negative control mech-
anisms. It should be noted that the action of stochastic
attrition would be highly nonlinear because once the
number of active channels falls below some critical level,
the remaining channels would be increasingly impacted
by the phenomenon.
C. Adaptation
Most single RyR channels studies have focused on
defining RyR behavior under steady-state conditions.
Gyo ¨ rke and Fill (112), using laser flash photolysis of caged Ca 2, were the first to apply fast trigger Ca 2
signals to single RyR2 channels in bilayers. They reported
that single RyR2 channels activated rapidly reaching P o values that were well above those predicted from previ-
ous steady-state studies. After an initial burst of single-
channel activity, P o spontaneously decayed with a time
constant of 1 s. Application of second Ca 2 stimulus
reactivated the apparently “inactivated” channels. This
suggested that the spontaneous decay observed after the
first Ca 2 stimulus was not due to a conventional absorb-
ing Ca 2 inactivation mechanism (81) because the chan-
nels were not in a refractory state. They proposed that the
spontaneous decay may be mediated by a different pro-cess they termed adaptation (112, 230).
The RyR2 channel adaptation proposal spurred an
active debate. One point of debate centered on the rela-
tively slow time course of adaptation (1 s). The point
was that adaptation might be too slow to be a negative
control mechanism that stabilizes the CICR process in
heart. Somewhat lessening this concern, Valdivia et al.
(339) demonstrated that the RyR2 channel adaptation
phenomenon was 10-fold faster in the presence of phys-
iological Mg2. Another point of debate was that not all
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investigators reported “adaptive behavior ” of single RyR2
channels. Although several groups reported that fast Ca 2
stimuli induced a transient burst of single RyR2 channel
activity (112, 159, 264, 283, 339), different groups inter-
preted their results in different ways. Still another point of
debate was centered on the nature of the Ca 2 stimulus
applied in the original adaptation study (112). It wassuggested that the fast Ca 2 overshoot at the leading edge
of the applied Ca 2 stimulus might generate the observed
adaptation phenomenon (154). As a result, the single
RyR2 channel data generated by the flash photolysis have
undergone an unusual level of scrutiny. It has endured,
has been frequently reviewed (86, 152, 288), and has
generated new insights into single RyR2 channel Ca 2
regulation. Details concerning the RyR2 adaptation de-
bate are discussed in section XVI.
We now know that the adaptation phenomenon is
due to a Ca 2- and time-dependent shift in the modal
gating behavior of single RyR2 channels (111). A fast-
sustained Ca 2 stimulus transiently drives the RyR2 chan-
nel into a high P o gating mode before it has time to
equilibrate (i.e., adapt) between all its different gating
modes. A specific kinetic scheme of RyR2 channel gating
has been developed describing this behavior (86). It is
now clear that Ca 2-dependent inactivation and adapta-
tion are not mutually exclusive mechanisms (as generally
thought). These two phenomena may simply be different
manifestations of a common underlying mechanism,
Ca 2- and time-dependent modal gating.
D. Activation-Dependent or “Fateful” Inactivation
Pizarro et al. (244) found that the global RyR1-medi-
ated Ca 2 release transient in frog skeletal muscle, trig-
gered by a maximally depolarizing stimulus, was inhibited
in proportion to the degree of Ca 2 release generated
during a prestimulation. This suggests that the RyR1 chan-
nels activated in the prestimulation were not available for
activation during the main stimulus. Thus preactivated
RyR1 channels appeared inactivated or refractory. These
authors proposed that RyR1 channel inactivation is
strictly and “fatefully” linked to their activation. In other
words, RyR1 channel inactivation is use dependent. Cer-
tain single-channel studies are consistent with this idea. Itis possible that a modal gating shift (i.e., high to low P omodes) may be a single-channel manifestation of fateful
inactivation (i.e., use dependence) observed in situ. Sev-
eral different groups have now reported modal gating
behavior of single RyR channels (8, 86, 111, 361).
E. Depletion/Luminal Ca2 Effects
When the SR is overloaded with Ca 2, periodic spon-
taneous Ca 2 waves can occur in heart muscle (304). It
appears that luminal Ca 2 levels modulate these RyR2-
mediated Ca 2 waves. The presence of a luminal Ca 2
regulation may also explain why a critical Ca 2 load must
be attained before Ca 2 can trigger SR Ca 2 release from
SR vesicle preparations (234–236). Several single RyR
channel studies have also addressed the possibility that a
luminal Ca 2
regulatory site exists (110, 341, 285, 333). Velez and Suarez-Isla (341) showed that RyR channel
activity changed as a function of luminal Ca 2 concentra -
tion and suggested this phenomenon was mediated by a
low-af finity Ca 2 binding site on the luminal side of the
channel. Later, two groups (110, 285) systematically in-
vestigated luminal RyR2 Ca 2 regulation and showed that
its extent depended on how the RyR2 channel was acti-
vated. For example, ATP-activated RyR2 channels were
more sensitive to luminal Ca 2 levels than Ca 2-activated
RyR2 channels.
The pertinent low-af finity luminal Ca 2 binding
site(s) may reside on the RyR channel or possibly on
another closely associated luminal protein. These sites
would need to sense changes in intra-SR Ca 2 levels in the
appropriate Ca 2 concentration range (0.2–20 mM). As SR
Ca 2 levels fall, the RyR channel would sense the reduced
SR Ca 2 level leading to a decrease in channel activity.
This would then represent an interesting form of RyR
negative control. There are caveats, however, in this lu-
minal Ca 2 story. Repetitive Ca 2 sparks should reduce
local SR Ca 2 load and thus downregulate RyR2 channel
activity. Yet, multiple Ca 2 sparks can occur frequently at
the same site in cells with little sign of progressive run-
down. This may suggest that SR Ca 2 levels either do not
change dramatically and/or that luminal Ca 2
may notimpact RyR2 channel activity dramatically. The spark
data, however, do not rule out the possibility that very
brief and highly localized luminal Ca 2 depletions govern
RyR2 channel function.
F. Allosteric or Coupled Gating
Recently, Stern et al. (305) evaluated several pub-
lished single RyR2 gating schemes and found that they all
generated unacceptable instability when applied in a
model of Ca 2 release local control. One reason for the
instability was the lack of a strong negative control mech-anism to minimize local regenerative Ca 2 release in clus-
ters of RyR2 channels. Another reason for the instability
was the relatively low cooperativity of the RyR2 channel
Ca 2 activation process. The low cooperativity does not
allow the RyR2 channels to adequately discriminate be-
tween trigger and background Ca 2 signals. Interestingly,
Stern et al. (305) suggested that RyR2-RyR2 allosteric
interactions could theoretically overcome these instabil-
ity problems. Recently, it has been reported that neigh-
boring RyR1 channels may be physically coupled by the
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FK506-binding protein (FKBP12) and consequently gate
in synchrony (188). Is this coupled RyR channel gating
operative under physiological conditions? The answer to
this question is not yet clear (19, 305). Other investigators
have not reported coupled RyR channel gating. Addition-
ally, certain thermodynamic considerations may also be a
problem (i.e., multiple large macromolecules operatingwith microsecond synchrony). Furthermore, removal of
FKBP12.6 from the RyR channel does not abolish Ca 2
sparks, but actually increases their frequency and dura-
tion (351). Although coupled RyR channel gating is an
interesting and provocative hypothesis, its existence and
nature still require verification.
XVI. ADAPTATION DEBATE
The Gyo ¨ rke and Fill (112) RyR2 adaptation hypothe-
sis was controversial. One concern was that the decrease
in RyR2 channel activity, interpreted as adaptation, mightreflect slow Ca 2 deactivation (154) following a short-
lived Ca 2 transient (i.e., the fast Ca 2 overshoot) at the
leading edge of the applied Ca 2 stimuli. This possibility
was experimentally addressed, and the data suggest that
the brief Ca 2 overshoot has little impact on the much
slower (i.e., 1,000-fold slower) adaptation phenomenon.
The data indicate that the impact of the fast Ca 2 over-
shoot is most likely limited to accelerating the initial
closed to open transition of the RyR2 channel (i.e., it
essentially supercharges the applied Ca 2 stimuli). A de-
tailed discussion of the Ca 2 overshoot concern is pre-
sented below after a detailed description of the Ca 2
overshoot itself. Another concern regarding the originaladaptation hypothesis (112) was that some investigators
did not report adaptive behavior of single RyR2 channels
but instead forwarded alternate interpretations (49, 112,
159, 264, 283, 339). Some of these different interpretations
are also presented below.
A. DM-nitrophen Ca2 Overshoot
DM-nitrophen is an ultraviolet-sensitive high-af finity
Ca 2 buffer that changes its Ca 2 af finity from 4.8 nM to
3 mM upon flash photolysis (74, 78, 194). Ca 2 liberation
by flash photolysis of DM-nitrophen (the caged Ca 2 com- pound used the RyR2 adaptation studies) generates a fast
Ca 2 overshoot (78, 154, 194, 372). It arises because flash
photolysis liberates Ca 2 faster than free DM-nitrophen
can bind Ca 2. Its characteristics and how its kinetics
vary with experimental conditions are not generally un-
derstood and often the basis of debate (112, 154, 372).
The fast Ca 2 overshoot is very fast compared with
kinetics of commonly used fluorescent dyes (78). Conse-
quently, the kinetic characteristics of the fast Ca 2 over-
shoot are necessarily based on numerical calculations
from the much slower fluorescence data even in the case
of the fastest fluorescent indicators (78). The parameters
estimated for the fast Ca 2 overshoot may then reflect
more the kinetic characteristics of the indicator than the
Ca 2 liberation kinetics of the caged Ca 2 compound.
There is no debate that DM-nitrophen photolysis by a
brief flash (50 ns long) liberates Ca 2
in 30 s (74, 78,194). However, there is disagreement about the Ca 2
association rate of DM-nitrophen. Zucker (372) suggested
a relatively slow association rate (1.5 106 M1s1), and
use of this value predicts that Ca 2 overshoots should last
several milliseconds. More recently, Escobar et al. (78)
showed that the association rate was substantially faster
(3 107 M1s1). This means that the fast Ca 2 over-
shoot is more than an order of magnitude faster (100 s
vs. 2 ms) than previously thought (154, 372).
The magnitude of the overshoot depends on the free
DM-nitrophen levels. At pCa 9, only 18% of unphotol-
ysed high-af finity DM-nitrophen will be bound to Ca 2.
Flash photolysis will liberate Ca 2 from Ca 2-bound DM-
nitrophen, but much of this Ca 2 will rebind to Ca 2-free
DM-nitrophen. At pCa 7, nearly 96% of unphotolysed high-
af finity DM-nitrophen will be bound to Ca 2. The same
flash will liberate much more Ca 2 from Ca 2-bound DM-
nitrophen, but there will be less Ca 2-free DM-nitrophen
to participate in rebinding. As a result, the Ca 2 overshoot
will be smaller and slower. At pCa 6, nearly all unphoto-
lysed high-af finity DM-nitrophen will be bound to Ca 2.
There will thus be no Ca 2 rebinding and consequently no
Ca 2 overshoot.
We estimate, based on the calculations presented in
previously works (78, 86), that the Ca 2
overshoot in theoriginal Gyo ¨ rke and Fill (112) study peaked at 30 – 60 M
and lasted 100–200 s. Gyo ¨ rke and Fill (112) used a
Ca 2 electrode to measure free Ca 2 concentrations be-
fore and after photolysis. Thus the properties of the fast
Ca 2 overshoot could not be measured. These kinetic
estimates predict that the fast Ca 2 overshoot was at least
three orders of magnitude faster than the observed adap-
tation phenomenon (112, 372).
B. Ca2 Overshoot Concern
The concern was that RyR2 adaptation might simplybe deactivation following the fast Ca 2 overshoot (154).
In other words, the Ca 2 activation sites will be rapidly
occupied during the overshoot. After the overshoot, oc-
cupancy of the site will fall and deactivate the channel.
The idea was that the RyR2 deactivation after the over-
shoot (lasting 200 s) generates the adaptation ( 1
s) phenomenon. If this were the case, then the deactiva-
tion of the RyR2 would need to be very slow. Is RyR2
Ca 2 deactivation so slow? The answer is no. The rate of
RyR2 channel deactivation in response to a fast Ca 2
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reduction has been experimentally defined (264, 340, 362),
and it is quite fast ( values 5 ms). Does the fast Ca 2
overshoot impact single RyR2 channel function? The an-
swer is yes. Zahradnı kova ´ et al. (362) using improved
recording conditions showed that the fast Ca 2 over-
shoot, albeit slightly smaller than those applied by Gyo ¨ rke
and Fill (112), can occasionally induce one or two brief opening events. Application of Ca 2 stimuli like those
applied by Gyo ¨ rke and Fill (112) (i.e., overshoot followed
by a sustained elevation), induce an entirely different
pattern of single RyR2 channel activity (including the
classical adaptation phenomenon). These data and more
recent single-channel modeling (86) suggest the impact of
the fast Ca 2 overshoot on single RyR2 channel function
may simply be to accelerate the initial opening transition.
Does the fast Ca 2 overshoot induce the adaptation
phenomenon? Few questions concerning single RyR
channel function have been debated more (86, 152, 288).
In principle, adaptation represents a slow transition of the
RyR channel from one state to another. There is circum-
stantial experimental evidence that suggests that this
slow transition is not simple Ca 2 deactivation following
the Ca 2 overshoot (154, 264, 340, 362). There are also
theoretical arguments that suggest that the slow transi-
tion is not induced by the fast Ca 2 overshoot. A specific
Markovian gating scheme, albeit still imperfect, has been
forwarded to describe RyR2 adaptation (see Fig. 3; Ref.
86). In this scheme, a fast Ca 2 overshoot is not required
to generate adaptation-like phenomenon. This scheme
suggests that RyR2 adaptation is due to a transient Ca 2-
dependent modal RyR2 gating shift (see sect. XVII and Fig.
3). There is, however, no experimental evidence directlyaddressing the salient point (i.e., demonstration that the
same channel response is elicited by a Ca 2 step with or
without the initial overshoot). Thus the question above
has not been definitively answered yet.
C. Differing Interpretations
Several studies have now defined the kinetics of sin-
gle RyR2 channel Ca 2 regulation (112, 113, 159, 264, 282,
283, 339, 340, 362). Although the data are generally con-
sistent, how the different data sets have been interpreted
varied a great deal. Some investigators choose to interprettheir data in terms of adaptation (112, 339, 340), whereas
others choose to interpret their data in terms of classical
Ca 2-dependent inactivation (159, 264, 283). Several fac-
tors contributed to the different interpretations. The var-
ious studies used very different techniques and conse-
quently applied very different types of Ca 2 stimuli. This
is important because even relatively small differences in
the characteristics of the Ca 2 stimulus can have a large
impact on single RyR2 channel function (81). For years,
the absence of adaptation following the mechanically in-
duced Ca 2 stimuli was construed as evidence against the
adaptation phenomenon. A great deal of attention was
focused on understanding how the Ca 2 overshoot could
generate “adaptation.” Attention is now more appropri-
ately focused on understanding how the different types of
Ca 2 stimuli may impact channel function. It is now clear
that the photolytic Ca 2 stimuli have a fast component
not present in the mechanical methods. Thus it is not
surprising that RyR channel behavior is different in the
different studies. The point is that the results of the flash
photolysis studies (112, 339, 340, 362) are not inherentlyat odds with those reported in the mechanical solution
change studies (159, 160, 264, 282, 283), and there is no
reason a priori to favor one type of study over another.
Each study should be evaluated on its own merits, for
each represents a heroic effort and makes a substantive
contribution. In our opinion, the differences in the Ca 2
stimuli make the data more (not less) interesting. The fast
photolytic Ca 2 stimuli are particularly interesting be-
cause they may mimic the Ca 2 changes that activate RyR
channels in cells.
FIG. 3. Markovian scheme of modal RyR2 channel gating. The chan-
nel recording is thought to reflect the fluctuation of the molecule be-tween four highlighted sets of states, including high and low open
probability ( P o) modes, to explain the bursting nature of the channel’sgating. Below are four simulated single channel records generated usingthis scheme. Each illustrates the predicted response to a fast steplikefree calcium change. The different colored regions of the recordings
correspond to the different colored sets of the gating scheme. [Adaptedfrom Zahradnı kova ´ and Zahradnı k (361).]
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XVII. MODAL GATING
Under steady-state conditions, single RyR2 channel
activity occurs in bursts. This means that channel open-
ings tend to be clustered in time instead of being ran-
domly distributed in time. The P o during a burst can be
either high or low (Fig. 4). The high P o and low P o burstsdo not occur randomly. Instead, the bursts tend to be
clustered as well. Trains of high P o bursts characterize a
high P o gating mode. Trains of low P o bursts characterize
a low P o gating mode. Extended periods of no openings
characterize a zero P o gating mode. Several different
groups have now reported the existence of modal RyR2
channel gating (8, 86, 261, 361).
How does modal RyR2 channel gating work? This is
still an open question. The identities, transitions, and
characteristics of the different RyR2 gating modes are
currently being defined. Preliminary interpretations sug-
gest that there is a dynamic equilibrium between at least
three different gating modes under steady-state condi-
tions (i.e., at a constant Ca 2 level). High, low, and zero P omodes are clearly evident in the steady-state activity of
single RyR2 channels (8, 86, 261, 361). There also appears
to be clear spontaneous shifts between the different gat-
ing modes. Simulated single-channel data are presented in
Figure 4 to illustrate some of these points. It is thought
that the dynamic Ca 2-dependent equilibrium between
gating modes is what generates the classical bell-shaped
steady-state Ca 2 dependence of the RyR2 channel (54,
161, 353).
An interesting thing happens when the Ca 2 level
changes suddenly. Small fast Ca 2 elevations (from a low
Ca 2 level) upset the existing dynamic equilibrium mo-
mentarily. The result is a transient change in channel
activity until a new equilibrium is reached. Specifically, it
has been argued that single RyR2 channel activity momen-
tarily rises to a P o level above that predicted by steady-
state measurements before it spontaneously decays (86).Channel activity decays as the channel reequilibrates be-
tween the gating modes at the new higher Ca 2 level (Fig.
4). Subsequent small fast Ca 2 elevations would induce
further transient modal gating shifts, inducing additional
transient episodes of high channel activity. This behavior
is reminiscent of the adaptation phenomenon. The mag-
nitude of the transient modal gating shift should depend
on the speed and magnitude of the Ca 2 stimulus. A slow
Ca 2 stimulus would not induce a dramatic modal gating
shift because mode reequilibration is time dependent (i.e.,
modes will reequilibrate during the slow stimulus). Large
Ca 2 stimuli should be less effective because at high Ca 2
levels the channel will begin to frequent the inactivated
state. Consequently, a slow large Ca 2 stimulus would not
induce a substantial transient modal gating shift but may
instead induce Ca 2-dependent inactivation.
Is this consistent with the experimental record? Ap-
plications of fast Ca 2 elevations do indeed induce super
steady-state P o levels that are followed by a spontaneous
decay in channel activity (112, 159, 339). Also, repeated
episodes of transient channel activity in response to mul-
tiple incremental Ca 2 elevations have been reported
(112). Experiments have also defined the dependence of
channel activation rate on stimulus speed (159, 264, 283)
FIG. 4. Modal gating of the RyR2 channel. The Mark-ovian scheme shown in Figure 3 was used here to simulate
the single-channel recordings shown. At the top, a seriesof high P o bursts or low P o bursts are marked. Trains of high or low P o bursts correspond to the high or low P ogating modes of the RyR2 channel. At any steady-statecalcium concentration, the RyR2 channel will spontane-ously shift between its different gating modes. A samplespontaneous modal gating shift (at pCa 5) is shown in the
middle panel. In this case, the channel shifted from thelow P
o mode to the high P
o mode and back. Shifts in model
RyR2 gating can also be evoked by fast calcium concen-tration changes. A sample evoked modal gating shift is
shown on the bottom panel. A fast calcium concentrationchange (pCa 7 to 6) was introduced at the arrow. Thechannel immediately shifts into the high P o mode for a
period of time before shifting again into the low P o mode.
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and clear refractory behavior at very high Ca 2 levels
(264). Thus the modal gating theory qualitatively pre-
sented above has some experimental support. It is clear,
however, that additional experimental results are needed.
For example, current modal gating theory does not yet
consider the potential impact of certain physiologically
important ligands (e.g., luminal Ca 2, cytosolic Mg2, and ATP).
Is modal RyR channel gating physiological? Modal
gating may reproduce and reconcile certain single RyR
channel phenomena in bilayers, but its physiological rel-
evance is questionable. In cells, the RyR channels gener-
ate intracellular Ca 2 signals that last less than 25 ms
(e.g., sparks). Studies of “steady-state” RyR behavior in
bilayers report that intermodal gating transitions occur
over seconds. From these data, it appears unlikely that
modal RyR gating is a physiologically relevant phenome-
non. Modal gating may simply be a biophysical ramifica-
tion of RyR channel Ca 2
regulatory complexity. Thisdoes not make it less important because understanding
the origin of modal gating may provide new insights into
RyR channel Ca 2 regulation.
XVIII. CONCLUDING REMARKS
The advent of in vitro single RyR channel recording
has provided an abundance of information. It has revealed
key properties of the RyR channel’s permeation pathway
and how (and if) particular ligands alter channel function.
Despite some amazing advances, many fundamental ques-tions about how the RyR channels are turned on and off
in vivo remain unanswered. For example, in vitro single
RyR2 channel recording has not definitively established
the identity of the mechanism(s) that terminate the SR
Ca 2 release process or revealed how movement of the
DHPR voltage sensor governs RyR1 channel activity.
Our understanding of single RyR channel regulation
is clearly incomplete. This impairs our understanding of
many intracellular calcium signaling phenomena. Long-
standing RyR channel mysteries remain and are certain to
motivate research long into the future.
We thank the reviewers for their meticulous revision of the
manuscript and for providing us with very constructive com-
ments. We also thank Dr. J. Ramos-Franco and M. Porta for
comments on the manuscript and A. Nani for technical help.
Research in the laboratory of M. Fill is supported by Na-
tional Heart, Lung, and Blood Institute Grant HL-57832. J. A.
Copello is supported by American Heart Association Grant
0130142N.
Address for reprint requests and other correspondence: M.
Fill, Dept. of Physiology, Loyola University Chicago, 2160 South
First Ave., Maywood, IL 60153.
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922 MICHAEL FILL AND JULIO A. COPELLO
Physiol Rev • VOL 82 • OCTOBER 2002 • www.prv.org
8/12/2019 Ryanodine Receptors 2
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doi:10.1152/physrev.00013.200282:893-922, 2002.Physiol RevMichael Fill and Julio A. CopelloRyanodine Receptor Calcium Release Channels
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