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POLYCYSTIC RENAL DISEASE
1 in 500 autopsies
1 in 3000 hospital admissions
Accounts for ≈10% of end-stage renal failure
Autosomal dominant inheritance
WILSON’S DISEASE
1 in 100 individuals carry mutation in ATP7B gene (Cu-ATPase)
1-4 per 100,000 people
Autosomal recessive inheritance
Neurological or psychiatric symptoms
Liver disease
Kaysar-Fleischer (KF) ring
CYSTIC FIBROSIS
1/2000 births in white Americans
Median age for survival late 30s
Autosomal recessive inheritance
HYDROPHOBICMOLECULES
SMALLUNCHARGED
POLARMOLECULES
LARGEUNCHARGED
POLARMOLECULES
IONS
OCONbenzene
2
2
2
H Oureaglycerol
2
glucosesucrose
H , NaHCO¯ , KCa , Cl¯Mg
+
3
2+
2+
+
+
syntheticlipid
bilayer
THE RELATIVE PERMEABILITY OF A SYNTHETIC LIPID BILAYER TO DIFFERENT MOLECULES
Intracellular Extracellular Concentration ConcentrationComponent (mM) (mM)
Cations Na 5-15 145 K 140 5 Mg 0.5 1-2 Ca 10-4 1-2 H 8 x 10-5 (pH 7.1) 4 x 10-5 (pH 7.4)
Anions Cl 5-15 110 Because the cell is electrically neutral the large deficit in intracellular anions reflects the fact that most cellular constituents are negatively charged. The concentrations for Mg and Ca are given for free ions.
COMPARISON OF ION CONCENTRATIONS INSIDE AND OUTSIDE A TYPICAL MAMMALIAN CELL
THE PROBLEM:A CELL
-
+
-
+
-
+ -
+
-
+
-
+-
+-
+
-
+
-
+
-+
- +
-
+
-
+
-
+
-
+
-
+
H O2
SOLUTIONS:
-+
+
-
+
-
+
-
+
-
+
-
-
+
H O2
-
+
+
-
+
-
+
-
+
-
+
-
-
+
- +
-
+
H O2
-
+
+
-
+
-
+
-
+
-
+
-
-
+
+
+
-
H O2
-
+
+
-
+
-
+
-
+
-
+
-
-
+
+
-
- +
-
+
-+
-+
- +
IONS
A Cross between Human Beings and Plants . . .
SCIENTISTS ON VERGE OF CREATING PLANT PEOPLE . . .
Bizarre Creatures Could do Anything You Want
Tuesday, July 1, 1980
Normal Died Asymptomatic Lethargic500
450
350
300139 baseline 119 in 2 h 122 in 3.5 d 99 in 16 d
Plasma Na concentration (mEq/l)
400
Bra
in w
ate
r (g
/10
0 g
dry
wt)
Woman drinks so much water she dies January 13, 2007
SACRAMENTO, California (AP) -- A woman who competed in a radio station's contest to see how much water she could drink without going to the bathroom died of water intoxication, the coroner's office said Saturday.
Jennifer Strange, 28, was found dead Friday in her suburba n Rancho Cordova home hours after taking part in the "Hold Your Wee for a Wii" contest in which KDND 107.9 promise d a Nintendo Wii video game system for the winner.
"She said to one of our supervisors that she was on her way home and her head was hurting her real bad," said Laura Rios, one of Strange's co-workers at Radiological Associates of Sacramento. "She was crying, and that was the last that anyone had heard from her."
Copyright 2007 The Associated Press. All rights reserved.This material may not be published, broadc ast, rewritten, or redistributed.
Simple DiffusionF
lux
[S]o
• Flux is proportional to external concentration
• Flux never saturates
PROTEIN MEDIATED MEMBRANE TRANSPORT
• PRIMARY ACTIVE
• SECONDARY ACTIVE TRANSPORT
• FACILITATED DIFFUSION
• ENDOCYTOSIS/TRANSCYTOSIS
Membrane Flux (moles of solute/sec)
• Simple Diffusion
• Carrier Mediated Transport• Facilitated Diffusion• Primary Active Transport• Secondary Active Transport
• Ion Channels
.
electrochemicalgradient
simplediffusion
channel-mediateddiffusion
carrier-mediateddiffusion
PASSIVE TRANSPORT(FACILITATED DIFFUSION)
ACTIVE TRANSPORT
transported molecule
lipidbilayer
TRANSPORT OF MOLECULES THROUGH MEMBRANES
CARRIER MEDIATED TRANSPORT
COUPLED TRANSPORT
UNIPORT
lipidbilayer
SYMPORT ORCOTRANSPORT
ANTIPORT ORCOUNTERTRANSPORT
Membrane Potential Review• The lipid bilayer is impermeable to ions and acts like an
electrical capacitor.
• Cells express ion channels, as well as pumps and exchangers, to equalize internal and external osmolarity.
• Cells are permeable to K and Cl but nearly impermeable to Na.
• Ions that are permeable will flow toward electrochemical equilibrium as given by the Nernst Equation.
Eion = (60 mV / z) * log ([ion]out / [ion]in) @ 30°C
• The Goldman-Hodgkin-Katz equation is used to calculate the steady-state resting potential in cells with significant relative permeability to sodium.
⎟⎟⎠
⎞⎜⎜⎝
⎛
∗+∗+∗∗+∗+∗
∗=outClinNainK
inCloutNaoutKm [Cl]P[Na]P[K]P
[Cl]P[Na]P[K]Plog60mVV
Structure of a Potassium Channel
Doyle et al., 1998
• Higher flux than predicted by solute permeability
• Flux saturates• Binding is selective
(D- versus L-forms)• Competition• Kinetics:
[S]o << KmM [S]
[S]o = Km M = Mmax / 2
[S]o >> Km M = Mmax
Carrier-Mediated Transport
[S]oKm
Mmax
0.5Flu
x
MEMBRANE ION TRANSPORT PROTEINS
inout
3Na
2K
ATP
ADP + Pi
Na,K-ATPase
2 major subunits
Mr=112kDß Mr≈60kD
,inhibited by ouabain,digoxin digitalis
, -H K ATPase
2subunitsMr=114kDß Mr≈60-85kD
inhibited by Schering28080, ,omeprazolelansoprazole
inout
H
K
ATP
ADP+P i
inout
2Ca
ATP
ADP+P i
-Ca ATPase
Mr=110kD
- SR Ca ATPase inhibited ,by thapsigargin
cyclopiazonic acid
- :Other P Type Ion Transport ATPases
-Cu ATPase-H ATPase-Cd ATPase
-K ATPase
1- :FoF Type ATPases
-H ATPase-K ATPase
EXCHANGERS/COUNTERTRANPORTERS:
Na/H Exchanger
Mr = 90 kD
inhibited by amiloride
inout
H
Na
Na/Ca Exchanger
Mr = 108 kD
inhibited by dichloro-benzamil, exchangerinhibitory peptide (XIP)
inout
Ca
3Na
Anion ExchangerCl/HCO3 Exchanger
Mr ≈ 102 kD
inhibited by DIDS, SITSphenyl isothiocyanate
inout
HCO3
Cl
COTRANSPORTERS:
Na,K,2Cl Cotransporter
Mr = 120 kD
inhibited by bumetanidefurosemide
Na,glucoseCotransporter
Mr = 73 kD
inhibited byphlorizin
inout
2Cl
Na,K
inout
glucose
2Na
dSCo/dt = k+ [S]o [C]o – k- [SC]o = 0 at equilibrium
k+ [S]o [C]o = k- [SC]o
k- / k+ = ([S]o [C]o)/[SC]o = Km [SC]o = ([S]o [C]o)/Km
Fractional Rate = M / Mmax = [SC]o / ([C]o + [SC]o)
M = Mmax / (1 + [C]o/[SC]o) = Mmax / (1 + Km/[S]o)
Transport Kinetics
So + Co SCo Si S = Solute C = Carrierk +
k -
Reversible Transport
Co Ci
So Si
SCo SCi
Mnet = Min – Mout =
Mmax 1 11 + Km / [S]o 1 + Km / [S]i
-( )
• Uses bidirectional, symmetric carrier proteins
• Flux is always in the directions you expect for simple diffusion
• Binding is equivalent on each side of the membrane
Examples include: Glucose Transporters (GLUT); Anion Exchanger; Organic Anion Transporters; Urea Transporters; Monocarboxylate (lactate) Transporters (MCTs); Amino Acid Transporters; Zn Transporters (ZIP)
Facilitated Diffusion
• Uses bidirectional, symmetric carrier proteins
• Flux is always in the directions you expect for simple diffusion
• Binding is equivalent on each side of the membrane
Facilitated Diffusion
Facilitated Diffusion: Band 3/AE1
Facilitated Diffusion: Band 3/AE1
Cytoskeletal/AE1 Interactions
Primary Active Transport: Driven by ATP
• Class P – all have a phosphorylated intermediate• Na,K-ATPase H,K-ATPase• Ca-ATPase• Cu-ATPase• H-ATPase • bacterial K-ATPase• Phospholipid Flippase
• Class V • H+ transport for intracellular organelles
• Class F• Synthesize ATP in mitochondria
Primary Active Transport: Na,K-ATPase
• 3 Na outward / 2 K inward / 1 ATP• Km values: Nain = 20 mM Kout = 2 mM• Inhibited by digitalis and ouabain• Palytoxin “opens” ion channel• 2 subunits, beta and alpha (the pump)• Two major conformations E1 & E2• Turnover = 300 Na+ / sec / pump site @ 37 °C
3 Na
2 K
ATP
ADP + Pi
Intracellular2K 3Na
ADP
E2.ATP.K2
E2.(K2)ATP
E2.(K2)
E2P.K2
3Na
E1P
E2.ATP
E1P.Na3
E1P.(Na3)
E2P.ADP.Na3E1.ATP.Na3
Pi
Extracellular2K
ATP
E2P
Na,K-ATPase Reaction Scheme
Membrane Transport and Cellular Functions that Depend on the Na,K-ATPase
COOH
SE
L KP
TYQDRVAPPGLTQIP
Q I Q K T E I S F R P N D P K S Y E A Y V L N I I R F L E K Y K D S AQ
K P C I I I K L N R V L G F K P K P P K N E S L E T Y P L T M KY
N
PNVLPVQCTGKRDEDKDKVGNIEYFGMGGFFY
T L D T E I R I E C K A Y G E N I G Y S E K D R F Q G R F D V K I E V K S
E G
G
N L
PLQYYPYYGK
K
NDESY
G Y K
L
250
LQP
Y L Q P L L A V Q F T
K
YGF
100
200
150
300
Y 50
NH2G
R
DK
Y
EPA
A
V
SE
HG
DK
K
K
S
AK
K
ER
DMD
E
LKK
E
VS
MD
DH
KL S L D
EL
HR
K
GTD
L
S
RGL
TP
A
RP
AE
IL
ARD
G
PQ
RL
T
PP
PT
TP
EW
V
K
FC
R
TNC
V
E
GT
ARG
IV
VY
T
G
DR
TV
M
GR
I
AT
LA
S
GLE
GG
QT
P
IAE
E
I
E
H
F
T
F
EPQ
T
RS
PD
F
T
NE
NP
LE
TR
N
IA
F
S
A
L H C F G
R
FI
D
E D P L LL N D V P F N V E D T D F Q F G E P F QD
I M S I L G V F C
K C R S
P
P
RA A V P A V A G I K V I M V T G D H P I T A A I G V G I I S
LY T
N
E G
YH
LR
SE E T M D K A D R P N V Q N V P I N L R A A I D
TE
N
T E I V F A R T S P Q Q K L I V E G C700
Q R
D
L
K
I
A
D D L L D S G H V V C A K
I D A K K L A P S D N V G D G T V A V IA
GQ
DF750
AT IV SV G
D V S K Q
GM A V G
IV G S A A D
N
M IL
T
S
G
EE
L
EPINS
COOH
L C
IGL YA
F
G
I
CT
T
D
LT V 800
L
L
I
D
TA
SR
E PP
E
DN
D
T
R 1000
F
L
LI I 950
Extracellular
Cytoplasmic
K
D
QL F G
G F S ML WL
I G A I
Y L G
V V L S
A V V
I I T G
C FS
YYQ
EA
KS
S
KI
M
E
S
FKN
M
VPQ
Q
A
L
VI
R
N
GE
KM
S
INA
E
D
V
VV
GD
L
VE
V KG
GD
RI
P
A
DL
R
II
S
AN
GC
KV
D
NS
S
L
G E S
I H LI T G
V A V FL G V
S F F IL S L
I L
EY T W L
E
A V I FI G I
I V A NL
V P E G
L L A T
V T V C
L T L TA
KR
MA
RK
N
N
CL
V
K
N
LEA
VE
TLG
STS
TI
CS
DK
TG
T
LT
E
QNR
M
T
VA
H
M
W
FD
N
QI
H E A D T T E Q S G V S F D K T S A T W F A L S R I A G L C N R A V F Q A N Q E N L P I L K R A VAGD
ASESLLKCIEVCCGSVMEMREKYTKIVEIPFNSTNKYQLSIHKNPNA
S
EP
K H L L V M K G A P E R I L D R C S S I L L H G K E Q P L D E E L K D A F Q N A Y L E L GGL
GERVL
G600
K A K
VE
650I
I
L
I
I
GL P L
P
AN
FI I
I
P F
M V P
A I S
L A Y
A
E
ESDI
MK
RQ
P RN
PK
TD
KLVN
ER
G 850
L I S MA Y
Q I G MI Q A
L G G FF T Y
F V IL
AENGFLP
FH
LL
GI R E T W D D R W I N D V E D S Y
GQQ 900
WT
YEQRKIVE
G LTE A
L A
F T C
H T A F
F V S
I V V V
Q W A
D L VI
CKTRRN
SV F
QQGMKN
K
FE
AF L S Y
C P G MG A A
L R MY
P L K
P W
W
TF C
A F P
Y S L
L I F
V D
EV
KL
II
RR
R
PG
G
W
V
EK
ETY
Y
100
150
200
250
300
350
400
450
500
550
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10
L ILF
Y V I F Y
GCLAGI F
M VI
TGI
LL
Q
T I
F
LGR
T
G
GSW
F
K
KDDMIFEDCGSMPSEPKERGEFNHERGERKVCRFKLDWLGNCS
MAR
G
K
AK
EE
G
SW
K
K
FI
W
N
SE
βKKE
NH2
GL
Y
52
K
Q
3/3IDENTICAL2/3IDENTICAL
NONE
4/4IDENTICAL3/4IDENTICAL
2/4IDENTICAL
NONE
1M
Amino Acid Homology Among the Na,K-ATPase Subunit Isoforms
QuickTime™ and aSorenson Video 3 decompressorare needed to see this picture.
The Na,K-ATPase As a Receptor For Signal Transduction
SR Ca-ATPase
FoF1 ATPase
QuickTime™ and aH.264 decompressor
are needed to see this picture.
Nat Commun. 2012 February 21; 3: 687
Experimental Evidence for Rotation
Secondary Active Transport
• Energy stored in the Na+ (or H +) gradient is used to power the transport of a variety of solutes
glucose, amino acids, ions and other molecules are pumped in (cotransport)
Ca2+ or H+ are pumped out 2 or 3 Na+ / 1 Ca2+ ; 1 Na+ / 1 H+
(countertransport)
• These transport proteins do not hydrolyze ATP directly; but they work at the expense of the ion gradient which must be maintained by an ATPase
Secondary Active Transport
• In humans over 40 families of Na coupled transporters
Examples include: Na+/H+ exchanger; Na+/Ca2+ exchanger; Na+/aspartate cotransporter; Na+/amino acid cotransporter; Na+/glucose cotransporter; Na+/urea cotransporter; Na+/PO4; cotransporter; (H+/Na+)/Zn2+ exchanger (ZnT)
Secondary Active Transport
• Energy stored in the Na+ gradient is used to power the transport of a variety of solutes
glucose, amino acids and other molecules are pumped in (cotransport)
Ca2+ or H+ are pumped out 2 or 3 Na+ / 1 Ca2+ ; 1 Na+ / 1 H+
(countertransport)
• These transport proteins do not hydrolyze ATP directly; but they work at the expense of the Na+ gradient which must be maintained by the Na,K-ATPase
Energy available from ATP H2OATP ADP + Pi
G = Gproducts – G reactants
Chemical Energy (G) = RT ln [C]
G = G° + 2.3 RT (log ([ADP] [Pi]) – log [ATP])
2.3 RT = 5.6 kiloJoules / mole @ 20° C
G° = -30 kiloJoules /mole @ 20°C, pH 7.0 and 1M [reactants] and [products] “Standard Conditions”
Energy Depends on Substrate Concentrations
G = -30 – 5.6 log [ATP] kJ / mole [ADP] [Pi]
The energy available per molecule of ATP depends on: [ATP] 4mM, [ADP] 400 µM, [Pi] 2 mM
per mole of ATP hydrolyzed:
G = -30 kJ – 5.6 kJ * log 4 x 10-3 2 x 10-3 * 4 x 10-4
= -30 kJ - 21 kJ = -51 kiloJoules per mole of ATP
Converting to approximately -530 meV/molecule of ATP
Energy in the Sodium Gradient
Consider Na+ movement from outside to inside:
G = Gproducts – Greactants = Ginside – Goutside
Gtotal = Gelectrical + Gchemical
Conditions for our sample calculation: Vm = -60 mV [Na+]out = 140 mM [Na+]in = 14 mM
and 2.3 RT = 60 meV / molecule
Energy in the Na Gradient: Electrical Term
Gelectrical = e mVin – e mVout
= +1e -60 mV – (+1e) 0 mV
= -60 meV
• negative sign means energy is released moving from outside to inside
• 60 meV is the energy required to move a charged ion (z=1) up a voltage gradient of 60 mV (assuming zero concentration gradient)
* *
* *
Energy in the Na Gradient: Chemical Term
Gchemical = 2.3 RT (log [Na+]in – log [Na+]out)
= 60 meV * (-1)
= -60 meV
• negative sign means energy is released moving from outside to inside
• 60 meV is the energy required to move a molecule up a 10 fold concentration gradient (true for an uncharged molecule or for a charged molecule when there is no voltage gradient)
Energy in the Na Gradient: Total
Gtotal = Gelectrical + Gchemical = -120 meV
• 120 milli-electron-Volts of energy would be required to pump a single Na+ ion out of the cell up a 10 fold concentration gradient and a 60 mV voltage gradient.
• Hydrolysis of a single ATP molecule can provide at least 500 meV of energy – enough to pump 4 Na+ ions.
• A single Na+ ion moving from outside to inside would be able to provide 120 meV of energy, which could be used to pump some other molecule, such as glucose, an amino acid, Ca2+ or H+ up a concentration gradient
Example: Na+/Ca2+ exchange
Compare the internal [Ca2+] for exchange ratios of
2 Na+ : 1 Ca2+ vs. 3 Na+ : 1 Ca2+
Vm = -60 mV, [Ca2+]out = 1.5 mM [Ca2+]in = ?
Ca2+ moves from inside to outside
G = Gproducts – Greactants = Goutside – Ginside
Gelectrical = (+2e) * (0 mV) – (+2e) * (-60 mV)
= +120 meV
Gchemical = 60 meV (log 1.5 – log ?)
Na+/Ca2+ exchange
2 Na+ 240 meV 240 = 120 + 60 log (1.5 / ?)120 / 60 = log (1.5 / ?) 102 = 1.5 / ? ? = 15 µM
3 Na+ 360 meV 360 = 120 + 60 log (1.5 / ?)240 / 60 = log (1.5 / ?) 104 = 1.5 / ? ? = 0.15 µM
Gtotal = GE + GC = 120 meV + 60 meV log (1.5 / ?)
Internal [Ca2+]can be reduced 100 fold lowerfor 3 Na : 1 Cavs 2 Na : 1 Ca
Structure of the Na/Ca Exchanger
Summary: Energetics
Transport Energetics • A molecule of ATP donates about 500
meV• It takes 60 meV to transport up a 60
mV electrical gradient• It takes 60 meV to transport up a 10
fold concentration gradient• A single sodium ion donates
approximately 120 meV
Summary: Membrane Flux (moles of solute/sec)
Simple Diffusion• Flux is directly proportional to external concentration• Flux never saturates
Carrier-Mediated Transport • Higher flux than predicted by solute permeability• Flux saturates• Binding is selective D- versus L-forms• Competition• Kinetics
Facilitated Diffusion• Uses bidirectional, symmetric carrier proteins• Flux is in the direction expected for simple diffusion• Binding is equivalent on each side of the membrane
Primary Active Transport – driven by ATP hydrolysis Secondary Active Transport – driven by ion gradients
Ion Channels
Transporters Regulated by Signaling Cascades
Na/H Exchangers
Na/Phosphate Cotransporter
Na/K/2Cl Cotransporter
Na/Cl Cotransporter
K/Cl Cotransporter
Na/Ca Exchanger
Na Channels
K Channels
Na,K-ATPase
H,K-ATPase
Unidirectional Transport Assays
Cells growingin multi-well plates
1. Cells washed in isotonic buffered solution
2. Required transport inhibitor(s) added
3. Flux medium containing radioactive isotope added
4. At required times flux medium rapidly removed and cells washed (3-4 x) in ice-cold isotonic saline
5. Final wash removed, cells lysed and radioactivity and protein content of samples determined
Calculations:
Specific Activity of medium:
Measure radioactivity in known volume of flux medium.
For example: For unidirectional uptake of Na into cells in medium containing:
50 mM Na100 mM choline Cl25 mM K-Hepes, pH 7.422Na (≈ 1 µCu/ml)
Measure radioactivity in 5 µl flux medium
Measure radioactivity and protein content in sample.
Determine Na influx using specific activity of mediumDetermine transport rate/protein content (Na uptake nmoles/µg protein/min)
Unidirectional Transport Assays
cpm (22Na) 1 L 1 mole cpm (22Na)
5 x 10-6 L 0.050 moles Na 109 nmoles nmoles NaX X =
THICK ASCENDING LIMB CELL
Na+
K+
Na+
K+
Na+
Lumen
Na+
K+
Na+K+
2Cl
K+ K+
Cl
Blood
HCO3
H2O
CO2
+
CA
H+
Na+
K+
H+Na+
BLOOD
"alkaline tide"
K+
H+
CO2
Cl
K+
HCO3
Cl
HCO3
Lumen
GASTRIC PARIETAL CELL
SMALL INTESTINAL CELL
Na+
K+
Na+K+
Cl 2Cl
Na+
H2O
K+cAMP
Lumen
