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OUTER PORE TOPOLOGY OF THE ECaC-TRPV5 CHANNEL BY CYSTEINE SCAN MUTAGENESIS

Running title: SCAM of the external pore residues in TRPV5

Yolaine Dodier, Umberto Banderali, Hélène Klein, Özlem Topalak, Omar Dafi , Manuel Simoes, Gérald Bernatchez, Rémy Sauvé, Lucie Parent*

Department of Physiology Membrane Protein Study Group (GEPROM)

Faculty of Medicine Université de Montréal

P.O. Box 6128, Downtown Station Montréal, Qué, H3C 3J7

Canada

* Corresponding author. Phone: (514) 343-6673 Fax: (514) 343-7146

e-mail: lucie.parent@umontreal.ca

Key words: Xenopus oocytes, calcium, kidney, cysteine, Site-directed mutagenesis, electrophysiology, MTS reagents, selectivity

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on November 20, 2003 as Manuscript M310534200 by guest on M

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ABSTRACT

ECaC-TRPV5 is a six-transmembrane domain calcium-selective ion channel that belongs to the large

family of TRP channels. The substituted cysteine accessibility method (SCAM) was used to map the

channel external vestibule and the pore region. Cysteine residues were introduced at 44 positions from the

end of S5 (E515) to the beginning of S6 (A560). Covalent modification by positively charged MTSET

applied from the external medium significantly inhibited whole-cell currents at 15/44 positions. Strongest

inhibition was observed in the S5-linker to pore region (L520C, G521C, and E522C) with either MTSET

or MTSES suggesting that these residues were accessible from the external medium. In contrast, the

pattern of covalent modification by MTSET for residues between P527 and I541 was compatible with the

presence of a α- helix. The absence of modification by the negatively charged MTSES in that region

suggests that the pore region has been optimized to favor the entrance of positively charged ions.

Cysteine mutants at positions -1, 0, +1, +2 around D542 (high-Ca2+ affinity site) were non-functional.

Whole-cell currents of cysteine mutants at +4 and +5 positions were however covalently inhibited by

external MTSET and MTSES. Altogether, the pattern of covalent modification by MTS reagents globally

supports a KcsA-homology based 3-D model whereby the external vestibule in ECaC-TRPV5

encompasses three structural domains consisting of a coiled structure (E515 to Y526) connected to a

small helical segment of 15 amino acids (527P-TALFSTFELFL-T539) followed by two distinct coiled

structures I540-P544 (selectivity filter) and A545-I557 before the beginning of S6.

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INTRODUCTION

The TRP ion channels form a large class of cationic channels that are related to the product of the

Drosophila TRP gene. TRP channels share a similar predicted topology of six transmembrane segments

in which the amino acids that link the fifth and sixth transmembrane domains line the pore region (1).

According to the recent IUPHAR classification of ion channels (2), the 21 members of the TRP family

can be divided by sequence homology into three subfamilies (3;4) as short (TRPCx), long or melastatin

(TRPMx), and osm-9-like or vanilloid-like (TRPVx) channels. The molecular domains that are mostly

conserved among TRP channels include part of the S6 segment, ankyrin repeats in the N-terminal, and a

"TRP domain" in the C-terminus (EWKFAR) (5), the latter being absent from TRPV channels. The

TRPC and TRPV proteins have two to four amino terminal ankyrin domains suggesting that these

proteins are coupled to the spectrin-based membrane cytoskeleton.

TRP channels vary significantly in their biophysical properties and gating mechanisms. In contrast to

other members of the TRP family, TRPV5 and TRPV6 channels show strong inward rectification,

exhibit anomalous mole-fraction effect, are activated by low [Ca2+]i and inactivated by higher [Ca2+]i (6-

8). TRPV5 and TRPV6 are also highly Ca2+-selective channels with PCa/PNa > 100. In particular,

ECaC- TRPV5 displays a high Ca2+ affinity with a Kd ≈ 2 µM (7) that is comparable to the Kd ≈ 1 µM

for voltage-dependent CaV channels (9). A single residue in the S5-S6 linker (D542) was found to

account for the high Ca2+ affinity of ECaC-TRPV5 (7). The absence of the aspartate residue at the

equivalent position in the pore region of TRPV1-4 channels might explain, together with the presence of

a lysine residue, the ≈ 20-fold lower Ca2+ selectivity of TRPV1-4 channels (10). TRPV5 and TRPV6

channels can also form homo- and hetero-tetramers suggesting that they are structurally and functionally

related (11).

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There is currently very little structural data available on the pore architecture of Ca2+-selective TRP

channels. It is possible that the four aspartate residues form an extracellular ion binding site as it has been

shown for the E71/D80 residues in the KscA crystal structure (12). It has also been proposed that the four

aspartate residues project in the pore lumen as it has been suggested with the four EEEE-residues locus

that accounts for the channel high Ca2+ -affinity (13) of Ca2+-selective CaV1.2 channels. In two landmark

studies, cysteine mutation of each of the four EEEE-residues locus of the CaV1.2 channel rendered the

channel susceptible to irreversible inhibition by external sulfhydryl modifiers, indicating that the side-

chain was covalently modified by the MTS reagent (14;15). Cysteine substitutions at positions

immediately adjacent to the EEEE locus (±1 positions) were also generally susceptible to sulfhydryl

modification. Sulfhydryl modifiers had lesser effects on channels substituted one position further from the

EEEE locus (±2 positions). These results suggested that the carboxylate-bearing side chains of the high-

affinity EEEE locus and their immediate neighbors were accessible from the water-filled extracellular

medium in CaV1.2 channels.

To examine the topology of the pore region and the external vestibule in TRPV channels, we undertook a

systematic analysis of pore residues accessibility using the substituted cysteine accessibility method

(SCAM)(16). Mutant channels were expressed in Xenopus oocytes and their covalent modifications by

externally applied membrane-impermeant methanethiosulfonate compounds of different charge and

cross-section were measured. Based on the reactivity / accessibility to external sulfhydryl reagents, we

report that some of the structural features of the bacterial KcsA and MthK channels, namely the coiled

region in the S5-pore linker (turret) and the pore α-helix of 15 residues that follows the turret and

precedes the selectivity filter, are conserved in TRPV5 channels. Furthermore, our results show that

T528, S532, E535, and T539 in the pore helix region are selectively accessible to positive MTS reagents

from the external medium suggesting that they constitute part of the external vestibule in ECaC-TRPV5.

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MATERIALS AND METHODS

Site-directed mutagenesis of the rabbit ECaC-TRPV5. The cDNAs coding for the wild-type ECaC-

TRPV5 (GenBank AJ133128) (17) and the wild-type CaT2-TRPV5 (GenBank AF209196) (18) were

obtained after reverse – transcription of rabbit distal tubule mRNA as reported before (7). ECaC-TRPV5

and CaT2-TRPV5 were subcloned into the pT7TS vector (generously provided by Dr Paul A. Krieg,

University of Texas) using exonuclease III (19) for optimal expression in Xenopus laevis oocytes. Point

mutations in ECaC-TRPV5 were performed with 39-mer synthetic oligos using the Quick-ChangeTM XL-

mutagenesis kit (Stratagene, LaJolla, CA). The C556S channel was used as a template for all cysteine

mutations (see Results) and oligos were carefully designed to preserve that mutation. The nucleotide

sequence of the S5-S6 linker including the background C556S mutation (over 600 bp) was bi-

directionally analyzed using automatic sequencing by BioST (Lachine, Qué). DNA constructs were

linearized at the 3' end by BamHI digestion. Run-off transcripts were prepared using methylated cap

analog m7G(5')ppp(5')G and T7 RNA polymerase with the mMessage mMachine® transcription kit

(Ambion, Austin, TX).

Expression of CaT2-TRPV5 wild-type, ECaC-TRPV5 wild-type, and mutants in Xenopus oocytes.

Female Xenopus laevis clawed frog (Nasco, Fort Atkinson, WI) were anesthetized by immersion in 0.1 %

tricaine or MS-222 (3-aminobenzoic acid ethyl ester, Sigma) for 15 minutes before surgery as detailed

before (7;20). cRNA was injected at a concentration of 0.46 to 4.6 ng per oocyte depending upon the

channel (wild-type or mutant) being expressed. With only 0.46 ng of RNA, large whole-cell inwardly

rectifying currents (≈ -50 µA) were routinely recorded with Li+ as the charge carrier (see below) for the

wild-type channel less than 24 hours after injection. ECaC-injected oocytes were incubated at 18°C in a

calcium-free and serum-free Barth's solution for 24-48 hrs before experiments.

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Whole-cell Recordings. Whole-cell currents were measured at room temperature with a two-electrode

voltage-clamp amplifier (OC-725, Warner Instruments) as described before(7). Voltage and current

electrodes (0.1-0.2 MΩ tip resistance) were filled with 3 M KCl; 1 mM EGTA; 10 mM HEPES (pH 7.4).

Instantaneous current – voltage relationships were measured using voltage ramps from +80 to –150 mV at

a rate of 0.575 mV/ms from a holding potential of –50 mV. Whole-cell current-voltage curves (I-V) were

measured under control conditions in the presence of the nominally calcium-free Li+ solution (in mM):

120 LiOH; 5 EGTA; 2 KOH; 20 HEPES titrated to pH 7.35 with methane sulfonic acid. Ca2+-free

solutions were used since ECaC-TRPV5 undergoes a near-irreversible inactivation in response to a steady

Ca2+ influx over a 5 to 10 min period (7;8). Oocytes were perfused by gravity flow at a rate of 10 ml/min

or 167 µl/s. Taking into account the volume of the experimental bath and the dead volume in the

perfusion line, the bath solution was completely exchanged within the first 30 s of perfusion. PClamp

software Clampex 8 (Axon Instruments, Foster City, CA) was used for on-line data acquisition and

analysis. Unless stated otherwise, data were sampled at 10 kHz and low pass filtered at 5 kHz using the

amplifier built-in filter.

The 44 cysteine mutants were systematically tested for inward rectification and high-Ca2+ affinity to

insure that the introduction of cysteine residues did not distort the channel structure. Whole-cell peak

currents measured at –150 mV varied from –10 to –30 µA for most cysteine mutants (see Table I for data

on the pore helix region). Out of the 44 mutants tested, only 14 cysteine mutations failed to express

whole-cell currents significantly larger than non-injected oocytes. Altogether, the non-functional mutants,

E515C, N518C, N519C, F523C, F531C, F534C, L536C, I541C, D542C, G543C, P544C, L551C, P552C,

and Y555C were tested in a minimum of 3 different series of oocyte injections over a 1-yr period using

RNA concentrations 10-times higher than for the wild-type channel for culture periods of 2 - 4 days. In

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addition, non-functional mutants were incubated for periods up to 3 hours with 5 mM DTT prior to

experiments to reduce eventual intra-disulfide bonds.

ECaC1 wild-type and mutant channel affinity for Ca2+ ions was assessed from the calcium block of

whole-cell Li+ currents as described previously (7;9;21). Ca(OH)2 was added to the solution to obtain the

desired level of free calcium. The stability constants used to calculate the free calcium concentration were

taken from Fabiato and Fabiato (22). Ca2+ block was reversible at > 90% in all experiments reported here.

Data were analyzed using Origin 6.1 (OriginLab Corporation, Northampton, MA) software. Results are

presented as mean ± S.E.M. Unpaired Students's t-test was used for statistical comparison.

Substituted cysteine accessibility method. MTS (MethaneThioSulfonate) reagents MTSEA ((2-

aminoethyl)methanethiosulfonate bromide), MTSET (2-(trimethylammonium)ethyl methanethiosulfonate

bromide), and MTSES (sodium (2-sulfonatoethyl) methanethiosulfonate) were purchased from Toronto

Research Chemicals (Toronto, Canada). Positively charged MTSEA and MTSET are both 1 nm-long but

differ substantially in surface area with diameters of 3.6Å (10Å2) and 5.8Å (26Å2 ) respectively (23).

MTSET and MTSES are predicted to be of similar size although they differ in charge. Because the MTS

reagents are rapidly hydrolyzed (5-10 min), they were always prepared fresh before use as described

before (16). MTSEA is positively charged but also potentially membrane permeant under certain

conditions (24) whereas MTSET (positively charged) and MTSES (negatively charged) are considered to

be membrane impermeant (24). Whole-cell current traces were routinely recorded using voltage ramps in

the presence of Li+ as the charge carrier (120 mM LiMeS + 5 mM EGTA) in the following sequence: 1)

under control conditions, 2) after the bath addition of 5 mM MTS reagent for 30 s, 3) after 5 min

perfusion with 5 mM MTS, and 4) after washing out the unreacted MTS reagent for 10 min with the

control solution. In addition of providing the MTS response over a wide range of potentials, voltage

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ramps allowed us to check for the presence of rectifying currents throughout the course of an experiment.

For most mutants, the gradual increase in non-specific currents or leaky currents could be immediately

assessed from the gradual disappearance of the trademark inward rectification of ECaC-TRPV5 currents.

Experiments where non-specific leaks developed during the experiment were simply discarded. The

reversibility of covalent bonds was tested with 5 mM BMS (bis(2-mercaptoethylsulfone)) (Calbiochem,

San Diego, CA) a water-soluble reagent considered to be a superior reducing agent than DTT

(dithiothreitol)(25;26). The percentage of currents remaining after MTS modification was computed at

Vm = -150 mV using [(Iwash)/(Ictrl)] x 100 where Iwash is the whole-cell peak current remaining after

MTS application and washout and Ictrl is the whole-cell peak current measured before MTS application.

For the figures, current traces were averaged from n ≤ 4 separate experiments and are shown as the mean

± S.E.M. Hence, the thickness of the traces actually reflects the experimental variability of the MTS

response. The standard errors tended to be smaller in the absence of functional modification. Inhibition

was considered “significant” at p < 0.01 .

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Computer-predicted structure and homology modeling of the pore region in ECaC-TRP5.

Sequence alignments were performed with the INSIGHTII HOMOLOGY module which integrated the

threading technique Profiles -3D developed in the laboratory of David Eisenberg (27). The analysis of the

3D scoring table led to the choice of the KcsA channel as a possible template to be used for ECaC-

TRPV5 homology modeling. The score was considerably lower for MthK and the alignment would

require the introduction of several gaps in the structure. Computer-based homology modeling was

performed with Modeller V6.2 (28) using the crystal coordinates of KcsA (PDB 1BL8) as a template and

involved the generation of 50 monomer models of the ECaC-TRPV5 channel pore. Energy minimization

was carried out with CHARMM. The dimer was obtained by superposing two ECaC monomers onto the

KcsA tetramer. The surface 3-D representation of the ECaC-TRPV5 was generated with the INSIGHTII

software (Accelrys, San Diego) as described elsewhere (16).

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RESULTS

Modification of wild-type TRPV5 channels by MTS reagents

To examine the topology of the external vestibule and pore region in TRPV channels, we undertook a

systematic analysis of pore residues accessibility using the substituted cysteine accessibility method

(SCAM) with hydrophilic MTS reagents (MTSEA, MTSET, MTSES). MTS reactivity of the wild-type

rabbit ECaC -TRPV5 (GenBank AJ133238) was investigated in the presence of Li+. As explained earlier,

whole-cell currents were measured in the absence of Ca2+ since ECaC undergoes Ca2+-dependent

inactivation over a 5- 10 min period in the presence of Ca2+ (8) whereas current levels of ECaC-TRPV5

and CaT1-TRPV6 are stable in Ca2+ -free solutions with either Na+ (8) or Li+ (7;29) as the charge carrier.

As seen, whole-cell currents measured under these conditions are strongly rectifying at positive voltages

(Fig 1A-B) (Table I). Perfusion with 5 mM MTSES for up to 5 min did not modify the whole-cell

currents through ECaC-TRPV5 (not shown). However, external application of 5 mM MTSEA (Fig 1A) or

5 mM MTSET (Fig 1B) significantly inhibited whole-cell currents between –50 and –150 mV. Washout

with the saline solution reversed the MTSEA-induced inhibition (Fig 1A) but did not affect the MTSET-

induced inhibition (Fig 1B) suggesting that the former inhibition was non-specific whereas the latter

inhibition was truly covalent. This conclusion was further supported by the application of the reducing

agent BMS that was found to fully reverse the MTSET-inhibition confirming that the MTS-induced

inhibition involved the formation of a disulfide bridge between the MTS reagent and the channel-protein

(Fig 1B).

In contrast to ECaC-TRPV5, the highly homologous CaT2-TRPV5 (GenBank AF209196) channel was

not irreversibly modified by either MTS reagent (Fig 1C-D) (Table I). As seen, whole-cell Li+ currents

through CaT2-TRPV5 rectified strongly at positive voltages. CaT2-TRPV5 also displayed an anomalous

mole fraction effect between Ca2+ and Li+ and external Ca2+ was found to inhibit whole-cell Li+ currents

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with a Kd = 2 ± 1 µM (4) which is comparable to the value reported for ECaC-TRPV5 (7). CaT2-TRPV5

was completely insensitive to MTSET (Fig 1D) and MTSES (not shown) whereas MTSEA inhibition

observed after a 5 min-exposure, was completely abolished upon the reagent washout (Fig 1C). A

comparison of their primary structure indicated that 11 out of the 15 cysteine residues of ECaC-TRPV5

were strictly conserved in CaT2-TRPV5 (Fig 2). Six conserved cysteine residues are located in putative

transmembrane segments S1, S4, and S5 whereas that the remaining five residues are located either in the

N-terminal (C70, C112, C172, and C213) or in the C-terminal (C619). The three non-conserved residues

namely C4, C270 and C653 are located either in the intracellular N- and C-termini where they should be

inaccessible to modification by external perfusion of membrane-impermeant reagents. The cysteine

residue at position 556 in the pore region of ECaC-TRPV5 corresponds to H549 of CaT2 where it could

potentially be accessible from the external medium.

The conservative mutation of the C556 residue to a serine resulted in an ECaC-TRPV5 channel

completely insensitive to MTSET (Fig 3B) and MTSES (not shown) as demonstrated by the current

traces superimposed under all experimental conditions. The smaller MTSEA, documented to be

somewhat membrane - permeant(24), caused current inhibition of whole-cell currents after a 5 min

perfusion period that did not persist upon extensive washout of the reagent (Fig 3A). The global time

course of MTS modification of C556S channels following a 10 min period was reported for MTSEA (Fig

3C) and MTSET (Fig 3D) at two voltages +20 mV and –100 mV. MTSEA steadily decreased currents

measured at –100 mV over that period but currents could be recovered at 92 ± 3 % (4) after washout of

the reagent (Fig 3C). Whole-cell currents at +20 mV were not affected indicating that the MTSEA-

induced modification did not affect the channel rectification. Longer incubation periods up to 20 min with

MTSET or MTSES did not significantly affect whole-cell currents (not shown). Hence C556S channels

did not undergo covalent modification by either MTSEA, MTSET, or MTSES. The key biophysical

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features of the wild-type channel namely the steep inward rectification and the high affinity for Ca2+ ions

(Table I) were preserved in the C556S channel. The C556S channel was thus used as the template channel

for the cysteine mutations in order to extend the characterization of the pore properties of the ECaC-

TRPV5 channel undertaken in a previous work (7). Cysteine mutations were introduced one by one into

the C556S channel in the S5-S6 linker region from D515 to M555. In all cases, whole-cell currents were

measured after 5 min- exposure to 5 mM MTS (MTSEA, MTSET, or MTSES). The MTS-induced

modification was reported after extensive washout of the reagent solution to insure that channel

modification did not result from non-specific effects.

External MTS reactivity of the S5 – to-pore linker region D515 –Y526

The MTS reactivity was first studied in the region referred to as the S5-to- pore-linker region spanning

11-amino acids from D515 to Y526. With the exception of L520C, all mutant channels in that region

featured the typical inward rectification and the high-Ca2+ affinity of TRPV5 channels. Three consecutive

mutant channels L520C, G521C, and E522C were strongly inhibited by positively and negatively charged

MTS. Inhibition was nearly completed within 30 s and persisted through extensive washout periods with

the control solution. Average I-V data are shown in Fig 4 for E522C. As seen, perfusion with 5 mM

BMS, a potent reducing agent almost completely restored whole-cell currents in MTSEA- and MTSET-

modified E522C channels confirming that the channel had formed a covalent disulfide bridge with the

MTS reagents. BMS nonetheless failed to restore under the same conditions the whole-cell currents of

MTSES-modified channels. Altogether, these data suggest that L520C, G521C, and E522C were readily

accessible from the aqueous external medium.

Such robust MTS reactivity was nonetheless limited to these three residues in that region. The SCAM

data for the S5 -pore linker are summarized in Fig 5. Nothing can be inferred from positions 515, 518,

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519 and 523 since E515C, N518C, N519C, and F523C mutants failed to express significant whole-cell

currents. Of the remaining mutants in that region, only D525C was covalently modified by the three MTS

reagents and this, to a moderate extent with residual currents ranging from 60 ± 7 % (5) for MTSEA (p <

0.01), and 61 ± 8 % (4) for MTSET (p < 0.01), to 77 ± 8 % (4) for MTSES (p < 0.1) as measured at –150

mV. Mutants P517C and S524C were only partially inhibited by MTSEA with residual currents of 64 ± 8

% (4) and 45 ± 10 % (3) respectively, suggesting a limited access to larger reagents at these positions.

SCAM analysis of the P527-I540 region

Cysteine substitution in the P527-I540 region resulted in 11 out of 14 channels with typical inward

rectification properties and high-Ca2+ affinity (Table I). Only two mutants, F531C and L536C failed to

express whole-cell currents larger than –1 µA at –150 mV. The overall pattern of MTS modification in

that region is summarized in Figure 6A. Covalent modification by positively charged MTSEA and

MTSET reagents resulted in a significant inhibition (p < 0.01) of whole-cell currents at positions T528C,

A529C, S532C, and E535C that persisted upon extensive washout (Table I). Average I-V data for T528C

(Fig 7A-C) and E535C (Fig 7B-D) are shown in the presence of MTSEA (Fig 7A-B) and MTSET (Fig

7C-D). As seen, MTSEA and MTSET inhibition of T528C currents occurred within the time frame of

bath perfusion (< 30 s). The E535C mutant channel was also significantly inhibited by MTSEA (Fig 7B)

but inhibition by MTSET (Fig 7D) was reduced considerably and required a longer perfusion period. It

follows that MTSET reactivity was strong within the first half of the region (528C, 529C, 532C, and

535C) but absent after position 535 whereas the smaller MTSEA reagent induced robust inhibition down

to position 539 (Table I). Indeed, F537C and T539C were functionally modified by MTSEA with

residual currents of 59 ± 4% (5) and 36 ± 8% (4) respectively, after a 5 min- perfusion period. Mutants

L538C and I540C were completely insensitive to modification by MTSEA and MTSET.

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Although residues T539 and I540 are located a few residues away from the D542 residue responsible for

the high-affinity Ca2+ binding site (7), the MTS response of either mutant was not altered when

experiments were conducted in the presence of 1 µM free Ca2+, the concentration required to inhibit 50%

of the whole-cell Li+ currents (results not shown). This suggests that Ca2+ binding / transit through the

channel did not significantly alter the pore structure to such an extent that it would modify the side-chain

accessibility to MTSEA and MTSET.

Finally, MTSES reactivity was absent in the entire P527-I540 region. A slight increase in whole-cell

currents was however observed at some positions upon washout of the unreacted MTSES reagent

although in both cases there was no discernible change in the whole-cell currents in the presence of

MTSES. The absence of MTSES-induced modification suggests the presence of an intrinsic electrostatic

field which would prevent negatively charged ions to penetrate into the pore.

Cysteine mutations of the I541-P544 region yielded non-functional channels

Cysteine substitutions of residues I541-P544 surrounding the high-affinity Ca2+ binding site located at

D542, namely I541C, D542C, G543C, and P544C all yielded non-functional channels. Although this

absence of functional expression could result from a dysfunction of the permeation pathway, mutations of

D542 to Ala (A), Gly (G), Glu (E), and Asn (N) in the wild-type ECaC-TRPV5 channels were shown to

produce functional channels with whole-cell currents exhibiting the typical inward rectification properties

(7).

External MTS reactivity in the pore- to- S6 linker region A545-A566

As shown in Figure 2, the last structural region studied spanned from A545 up to A566 in the S6

segment. Figure 6B shows the histogram summarizing the MTS data for the 11 cysteine mutations in the

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pore-to-S6 linker (A545C to C556). Three consecutive positions in that region (A545C, N546C, and

Y547C) produced whole-cell currents with milder rectification properties. Five positions (545C, 546C,

547C, 549C, 550C) out of 11 positions in that region reacted strongly with external MTSEA and MTSET

with MTSET-modification resulting in residual currents of 39 ± 5 % (8) (p < 0.001); 47 ± 7 % (8) (p <

0.001); 34 ± 6 % (4) (p < 0.001); 53 ± 5 % (4) (p < 0.001); and 63 ± 2 % (4) (p < 0.01) at –150 mV

respectively. Furthermore, the negatively charged MTSES inhibited 545C, 546C, and 549C channels at p

< 0.001 suggesting that access to these residues was not determined by the charge of the reagent. This

region also included a series of mutants that were either non-functional (L551C, P552C, and Y555C) or

completely insensitive to MTS modification (S548C and M554C) whereas F553C was only partially

inhibited by external MTSEA with residual currents of 53 ± 7 % (5) (p < 0.01) measured at –150 mV.

The external accessibility of S6 residues was tested intermittently at positions A560, A561, A563, and

A566. The A563C channel was non-functional whereas the A560C, A561C and A566C channels

remained completely insensitive to modification by MTSET and MTSES (results not shown) but A560C

was moderately inhibited by MTSEA with residual currents of 67 ± 8% (7) as measured at –150 mV.

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DISCUSSION

A systematic analysis of some of the structural features of the external vestibule of ECaC-TRPV5 was

performed using the substituted cysteine accessibility method (SCAM). Positively charged MTS reagents

were generally the most reactive as expected for cation selective channels with MTSEA ≥ MTSET >>

MTSES. MTS-induced inhibition of ECaC-TRPV5 was reported after a 5 min-exposure period which is

10 times larger than the time constants reported for the rate of MTS modification in other ion channels

(30;31). Under these conditions, inhibition was not significantly voltage-dependent between –50 and –

150 mV. Given that it is the rate rather than the level of inhibition that is usually influenced by the

membrane electrical field (32), this observation suggests that channel inhibition had reached a steady

state (33). The majority of cysteine mutants preserved the robust I-V rectification and the high-Ca2+

affinity that are typical features of ECaC-TRPV5. Nonetheless four (4) mutant channels (L520C, A545C,

N546C, and Y547C) lacked both properties suggesting that the two molecular mechanisms could be

linked in TRPV5 as it has been inferred in TRPV6 (34).

Homology modeling of the S5-pore-S6 region of ECaC-TRPV5

To establish a structural correspondence with crystallized K+ channels, the S5-pore-S6 region of ECaC-

TRPV5 was analyzed using the structure-based threading PROFILES-3D method (INSIGHT II) (Fig 8).

Although there is basically no homology between ECaC-TRPV5 and KcsA at the primary sequence level,

the scoring table generated by PROFILES-3D revealed that the pore region of ECaC-TRPV5 is

structurally compatible with the K + channel KcsA which can thus be used as a template for homology

modeling. The resulting sequence alignment as well as 3-D representations of the pore region are shown

in Figs 8-9. The structure prediction globally supports a model whereby two helical regions comprising

the transmembrane S5 and S6 segments encompass a “pore domain” consisting of a coiled structure

(E515 to Y526) connected to a small helical segment of 15 amino acids (527P-TALFSTFELFL-T539)

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which in turn is attached to two coiled domains, the first one (I540-P544) containing the high-Ca2+

affinity D542 residue (7) and the second one extending from A545 to I557. The PROFILES- 3D -based

alignment establishes a structural correspondence between D542 in ECaC-TRPV5 and Y78 in the GYGD

signature sequence known to contribute to the S0 - S1 K+ external binding site (35) in the KscA crystal

structure obtained at 2.1Å resolution (12) thus positioning the high-affinity Ca2+ binding site at the

entrance of the external vestibule. As seen in Fig 9A, the selectivity filter between I540 and P544 could

behave as an extended beta strand or a coil region, both structures being compatible with the X-ray

structure of the selectivity filter or GYGD region in KcsA and MthK channels. This region is followed by

a coiled structure between A545 and I557 predicted to be longer than in KcsA. The 3-D-based structure

alignment finally suggests that the S6 α-helix should start at T558 whereas the current secondary

structure model (17) based on hydrophobicity plots predicts S6 should begin at C556.

The surface 3-D representation (Fig 9B) of the model ECaC-TRPV5 channel displays a comprehensive

color-coded picture of the residues modified by MTSET and their relative accessibility from the aqueous

medium. Residues which displayed the strongest MTSET reactivity are colored in red whereas partial

inhibitions are shown in magenta. Yellow-colored residues formed non-functional cysteine channels.

Residues that failed to react with MTSET are presented in blue. As seen, residues which displayed the

strongest reactivity appear to be located at the external interface and surround the pore area. Residues that

caused a partial inhibition (magenta) or failed to inhibit ion fluxes appear to be positioned further away

from the pore. Finally cysteine mutations that yielded non-functional channels are mostly found in the

selectivity filter or close to the pore helix region. It is however important to bear in mind that the

implication of nonexistent or partial functional modification cannot be immediately translated into

structural information. The absence of modification (residues shown in blue in Fig 9B) could result either

from the inaccessibility of the residue or else from a “silent” covalent modification that does not affect

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ion fluxes. Partial inhibitions could be produced by the limited inaccessibility of the residue or from a

decrease in the channel single-channel conductance coupled with an increase in the channel mean open

time (16) whereby the MTS reagent behaves like an open-channel blocker. Despite these limitations, the

surface 3-D representation globally supports our SCAM data to the extent that the strong reactivity of

some residues was correlated with their accessibility from the external medium whereas non-reactive

residues appear to be buried within the protein.

The D515-Y526 region could form a coiled structure in ECaC-TRPV5

The 3-D model obtained by homology (Fig 9A) predicts that the region spanning 11-amino acids from

D515 to Y526 in ECaC-TRPV5 should form a coiled structure. This region projects in the aqueous

external medium and is called the “turret” in the crystal structure of KcsA (36) , MthK (37), and

KirBac1.1 (38) channels. The residues located in such a structure are expected to be readily accessible

without any apparent periodicity in MTS-induced modification. The robust reactivity of three consecutive

residues in that region (L520C, G521C, and E522C) argues for the presence of a coiled region that is

easily accessible from the aqueous external medium. The SCAM data for the remaining S5-pore linker

residues showed however milder reactivity for since only D525C was moderately modified by the three

MTS reagents. Overall, the strong reactivity of three consecutive residues irrespectively of the charge of

the MTS reagent agree with the surface representation of the S5-pore-S6 region as these residues

correspond to the red-colored spots surrounding the channel pore (Fig 9B).

The pore helix in ECaC-TRPV5

The structural region in ECaC-TRPV5 spanning from P527 to T539 could correspond to the “pore helix”

brought to light by the X-ray structures of the bacterial KcsA (12;36), MthK (37), and KirBaC1.1 (38)

channels. It is assumed that the pore helix provides a structural support to the selectivity filter. Its net

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negative dipole should contribute to the stabilization of K+ in the water-filled central cavity of the closed

KcsA channel (39) and could account for charge selectivity in Ca2+-permeable glutamate receptors

AMPAR (40). In the 3-D model obtained by homology (Fig 9A), the side-chains of hydrophilic residues

T528, S532, E535, and T539 are projecting toward the selectivity filter. This orientation is compatible

with the observation that these hydrophilic positions were covalently modified by MTSEA and MTSET.

In addition, the location of the hydrophilic T533 residue could also explain its relative insensitivity to

MTSET and MTSEA. A529C is the only residue that was covalently modified by MTSEA and MTSET

despite being apparently inaccessible in the 3-D model. It could be speculated that the relative

accessibility of A529 derives from its relative proximity to the external medium being located at the same

height as D542. The 3-D model patterned after the dimensions of KcsA should also be refined to take into

account the observation that MTSET (26Å2) was able to reach position E535C whereas MTSEA (10Å2)

could go further down to T539C at the junction where the pore helix finishes and the selectivity filter

starts suggesting that the pore region is larger in ECaC-TRPV5 than KcsA. Finally, the 3-D model

suggests that the access to the pore has been designed to select positively charged ions. Given that the

side-chains of E535 and D542 appear to project in the same direction, we can speculate that these

negatively charged residues provide together a potent negative field that could prevent MTSES to reach

its targeted cysteine residue in the pore helix.

The structural features of the pore-to-S6 linker region in ECaC-TRPV5

The selectivity filter is predicted to encompass I540 to P544, a region that was devoid of functional

cysteine mutant channels and shown in yellow in Fig 9B. Furthermore, the P544 to I557 region that links

the selectivity filter to S6 is predicted to form a coiled structure (Fig 9A) considerably longer than the

equivalent region in the KcsA. The robust MTSEA and MTSET reactivity of the first three consecutive

residues (A545C, N546C, Y547C) is indeed compatible with the presence of a coiled structure.

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Furthermore, A545C, N546C, and V549C channels were significantly inhibited by the negatively charged

MTSES indicating that access to these residues was not limited as we observed in the pore helix region.

The SCAM data were not however helpful in determining the beginning of the α-helix in S6 since most

mutants after D550 were either non-functional (L551C, P552C, and Y555C) or else non-reactive

(M554C). The C556 residue found to confer high MTSET-reactivity to the wild-type channel is found at

the end of this coiled structure (Fig 9A) where it could be accessible from the extracellular medium. We

can only speculate that MTSEA was too small to block efficiently ion fluxes through the selectivity filter

since the dimensions of the 3-D representation are very approximate. Finally, the A560C, A561C, and

A566C residues present in the S6 segment were found to be completely insensitive to modification by

external MTSET and MTSES reagents whereas A563C was non-functional. As predicted by the 3-D

model (Fig 9B), these hydrophobic residues should be buried within the channel.

Comparison with the SCAM analysis of models K+ channels

There exists little data on the cysteine reactivity/accessibility of the external vestibule of crystallized K+

channels (KcsA, MthK, KirBac1.1) besides the observation that Y82C located upstream to the GYGD

(77-80) signature sequence, can be functionally modified by external MTSET in the KcsA channel (41).

SCAM was used to investigate the structural features of the extra-long extracellular S5-P linker that is

absent from ECaC-TRPV5 channels and was also used to study the external vestibule of voltage-gated

KV2.1 (42) channels. In this last case, cysteine-substituted residues in the I369-G377 region that includes

the lower part of the pore helix and the selectivity filter were either non-functional or insensitive to

external MTS modification (42) just as we have shown in ECaC-TRPV5. The alignment produced by

PROFILES 3-D would correlate A545C in ECaC-TRPV5 with Y380C in Kv2.1 (42). These residues

were shown to be functionally modified by MTSEA, MTSET, and MTSES in both channels. T539 in

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ECaC-TRPV5 would also be aligned with T373 in Kv2.1. Since T373C in Kv2.1 was unaffected by

MTSEA (42) whereas T539 in ECaC-TRPV5 was significantly inhibited by MTSEA, it is suggested that

the pore region is wider in ECaC-TRPV5 than in K+ channels. Definite answers to these questions will

await the X-ray crystal structure of TRPV channels.

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ACKNOWLEDGMENTS

We are deeply indebted to Dr George Chandy for critical reading of the manuscript and to Dr Benoit

Roux for stimulating discussions. We thank Julie Verner for oocyte culture; Michel Brunette for technical

assistance; and Claude Gauthier for artwork. L.P. is a Senior scholar from the "Fonds de la Recherche en

Santé du Québec". We acknowledge the support of the Kidney Foundation of Canada and the Canadian

Institutes of Health Research (MOP 13390 and MMA 62995).

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Table I. Covalent modification of ECaC-TRPV5 mutants in the pore helix region

% Control current at –150 mV Channel Mutants

I-V Rect.

Peak current

(µA) MTSEA MTSET MTSES 1 µM Ca2+

ECaC –TRPV5 Yes -19 ± 2 (32) 78 ± 7 (7) 14 ± 2 (4) 86 ± 2 (7) 35 ± 2 (3)

C556S Yes -23 ± 3 (21) 90 ± 18 (9) 92 ± 3 (5) 96 ± 4 (4) 52 ± 7 (3)

CaT2 - TRPV5 Yes - 8 ± 3 (14) 90 ± 11 (3) 96 ± 4 (4) 112 ± 17 (3) 28 ± 7 (4)

C556S + P527C Yes -25 ± 4 (13) 86 ± 5 (3) 97 ± 7 (3) 106 ± 10 (4) 62 ± 15 (3)

C556S + T528C Yes -33 ± 22 (24) 45 ± 6 (4) 45 ± 11 (3) 101 ± 7 (10) 64 ± 6 (7)

C556S + A529C Yes -15 ± 3 (17) 13 ± 3 (3) 48 ± 7 (3) 78 ± 3 (4) 58 ± 4 (7)

C556S + L530C Yes -16 ± 2 (15) 66 ± 5 (4) 96 ± 2 (3) 91 ± 3 (3) 51 ± 2 (5)

C556S + F531C N/E N/E N/E N/E N/E N/E

C556S + S532C Yes -26 ± 2 (31) 57 ± 7 (4) 64 ± 3 (4) 96 ± 9 (5) 62 ± 1 (3)

C556S + T533C Yes -19 ± (32) 58 ± 6 (11) 100 ± 5 (10) 81 ± 7 (4) 57 ± 2 (7)

C556S + F534C N/E N/E N/E N/E N/E N/E

C556S + E535C Yes -25 ± 2 (30) 36 ± 6 (5) 69 ± 3 (3) 90 ± 8 (4) 78 ± 6 (3)

C556S + L536C N/E N/E N/E N/E N/E N/E

C556S + F537C Yes -25 ± 2 (23) 59 ± 4 (5) 87 ± 5 (5) 87 ± 5 (5) 60 ± 4 (8)

C556S + L538C Yes -28 ± 4 (22) 104 ± 11 (4) 106 ± 13 (5) 105 ± 4 (5) 45 ± 3 (8)

C556S + T539C Yes -24 ± 1 (18) 36 ± 8 (4) 93 ± 3 (4) 102 ± 4 (3) 52 ± 5 (4)

C556S + I540C Yes -30 ± 3 (28) 108 ± 6 (9) 109 ± 7 (6) 100 ± 7 (9) 80 ± 1 (4)

TABLE I. Biophysical properties and covalent modification of wild-type TRPV5 channels and mutants in the putative pore helix region. Most mutant channels displayed the strong inward rectification of wild-type TRPV5 channels. Peak currents were measured in the presence of the nominally Ca2+-free Li+ solution. The percentage of control currents remaining after external MTS modification with 5 mM reagent was computed after washout of the unreacted reagent (see Materials and Methods). A value of 100 means there was no effect. Mutant channels preserved the high- Ca2+affinity as shown by the percentage of whole-cell currents measured after exposure to 1 µM free-Ca2+. Data were computed at Vm =-150 mV and are shown as mean ± S.E.M with the number n of experiments in parentheses.

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FIGURE LEGENDS. Figure 1. MTS reactivity of the wild-type ECaC- and CaT2-TRPV5 channel. Whole-cell currents

through ECaC- and CaT2- TRPV5 display a strong rectification at positive voltages under control

conditions. A-B. Bath perfusion with 5 mM MTSEA (A) or 5 mM MTSET (B) decreased currents by 30-

50% within the first 30 s of perfusion (not shown) whereas further perfusion up to 5 min (2-light gray

line) nearly abolished them. Only the MTSET-induced inhibition persisted after washout with the saline

solution (3-gray). Perfusion with 5 mM BMS (4-dark gray line) reversed the MTSET-induced inhibition

as seen with the overlapping 1-4 current traces indicating the covalent nature of the modification. Bath

perfusion with 5 mM MTSES did not affect whole-cell currents (not shown). C-D. In contrast, the wild-

type CaT2-TRPV5 channel was not covalently modified by MTS reagents. 5 min -perfusion with 5 mM

MTSEA (C) but not 5 mM MTSET (D) or 5 mM MTSES (not shown) partially inhibited whole-cell

currents (2 - light gray line). Washout with the saline solution to remove unreacted MTSEA fully restored

whole-cell currents indicating the non-specific nature of the inhibition. Whole-cell currents were

measured using a 450-ms ramp protocol from +80 to –150 mV in the presence of the 120 mM Li + 0 Ca2+

solution. Currents were normalized to the peak currents measured at –150 mV under control conditions

and reported as the mean current trace ± S.E.M (n ≥ 4). See Table I for complete details.

Figure 2. Putative secondary structure of the epithelial ECaC1-TRPV5 Ca2+ channel. The deduced

primary sequences of ECaC1 (GenBank AJ133128) and CaT-2 (GenBank AF209196) are highly

homologous with 84% overall identity. The 15 endogenous cysteine residues of ECaC1-TRPV5 are

shown as circles with the empty circles showing residues conserved between ECaC and CaT2 whereas

filled circles highlight cysteine residues absent in CaT2. The insert underscores the pore region in the S5-

S6 linker region with 46 out the 52 amino acids being strictly conserved between the 2 channels in that

region. A single cysteine residue (shaded box) differs in that region, the C556 in ECaC1 that is aligned

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with H549 in CaT2. The residue D542 previously identified as the key molecular determinant of high

Ca2+ affinity in ECaC1-TRPV5 is shown with an asterisk *.

Figure 3. The endogenous cysteine at position 556 accounts for the MTS reactivity of the wild-type

ECaC-TRPV5. A-B. C556S currents display the trademark inward rectification of TRPV5 wild-type

channels and the high-Ca2+ affinity (see Table I for details). Whole-cell currents remaining after 5 mM –

exposure with MTSEA (A) or MTSET (B) were reported as mean currents ± S.E.M. MTSEA-induced

inhibition was fully reversed by washout with the saline solution indicating the non-covalent nature of the

MTSEA inhibition. As seen, the C556S mutation eliminated the covalent inhibition by MTSET.

Experimental conditions were described in figure 2. C-D. Time course of MTS-modification in C556S

channels was measured at +20 mV (empty circles) and at –100 mV (filled circles) every 30 s up to 10 min

perfusion with 5 mM MTSEA (C), 5 mM MTSET (D), and 5 mM MTSES (not shown) followed by a 15

min washout with the saline 120 Li+ solution. Perfusion with MTSEA induced a steady decline in

currents that was however washed out at ≥ 90% by the saline solution to a level that is comparable to the

level reached under the same conditions with MTSET or MTSES perfusion.

Figure 4. MTSEA- and MTSET-induced inhibition of E522C was reversed by BMS. The current

traces of E522C were measured in the 120 Li + 0 Ca2+ solution and are shown as mean data ± S.E.M.

before and after perfusion of MTSEA (A), MTSET (B) and MTSES (C) reagents. Experimental

conditions were described earlier. Whole-cell current traces were measured under control conditions (1-

black line); after 30 s perfusion with 5 mM MTS (2-light gray line); after washout of the unreacted MTS

(3-gray line); and after perfusion with 5 mM BMS (4-dark gray line). Whole-cell currents through E522C

were rapidly and nearly completed inhibited by perfusion with MTS reagents. MTSEA- and MTSET-

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induced inhibition but not MTSES-inhibition was reversed by perfusion of 5 mM BMS confirming that

the inhibition involved the formation of a disulfide bond.

Figure 5. Summary of the SCAM results of the turret region in the ECaC-TRPV5 channel. Effects

of MTS reagents on the Cys mutants spanning the E515 to Y526 region in the ECaC-TRPV5 channel.

Mutants were perfused with 5 mM of the MTS solution for 5 min in the 120 Li MeS (5 EGTA) solution

using the protocol described previously. The histogram reports the % of whole-cell currents remaining

after washout of the MTS reagents with the saline solution such that a ratio of 100 indicates that whole-

cell currents were not modified by MTS reagents as compared to control current traces. Whole-cell

currents through E515C, N518C, N519C, and F523C were not significantly different than currents

measured in non-injected oocytes In some cases, whole-cell currents increased upon washout of the MTS

reagent but this increase was not statistically significant (p > 0.01). MTSEA, MTSET, and MTSES

rapidly inhibited whole-cell currents for L520C, G521C, and E522C at p < 0.001 (**). MTSEA inhibited

whole-cell Li currents of P517C and S524C at p < 0.01 (data not shown).

Figure 6. A. Histogram of the SCAM data for the pore helix region in the ECaC-TRPV5 channel.

Effects of MTS reagents on the Cys mutants spanning the P527 to I541 region in the ECaC-TRPV5

channel. Mutants were perfused with 5 mM of the MTS solution for 5 min using the protocol described

previously. The histogram reports the % of whole-cell currents remaining after washout of the MTS

reagents with the saline solution such that a ratio of 100 indicates that whole-cell currents were not

modified by MTS reagents as compared to control current traces. Whole-cell currents through F531C,

F534C, and L536C were not significantly different than currents measured in non-injected oocytes In

some cases (P527C, L538C, and I540C), whole-cell currents increased upon washout of the MTS reagent

but this increase was not statistically significant (p > 0.01). MTSES did not significantly modify any

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mutant in this region. Inhibition by MTSET was significant at p < 0.001 for T528C and A529C and at p <

0.01 for S532C and E535C. Inhibition by MTSEA was significant at p < 0.001 for A529C and T539C

while significant at p < 0.01 for T528C, S532C, E535C, and F537C (Table I). B. Histogram of the

SCAM data for the pore to S6 linker region. Effects of MTS reagents on the Cys mutants spanning the

A545 to C556 region. The C556 channel is the wild-type channel (fig 1).Whole-cell currents for L551C,

P552C, and Y555C were not significantly different than currents measured in non-injected oocytes.

Strong reactivity for MTSEA and MTSET was observed at the beginning of this region with A545C,

N546C, Y547C, and V549C being significantly inhibited by MTSEA and MTSET at p < 0.001. In

addition, MTSES significantly inhibited A545C, N546C, and V549C suggesting that charge was not

critical in that region. D550C was inhibited by MTSEA at p < 0.001 and MTSET at p < 0.01. F553C was

inhibited by MTSEA at p < 0.01. C556S was inhibited by MTSET at p < 0.001. Mutants S548C and

M554C were not significantly modified by either MTS reagent. * indicates p < 0.01 and ** indicates p <

0.001.

Figure 7. SCAM analysis of the upper pore helix region in the ECaC-TRPV5 channel. The current

traces of T528C (A, C) and E535C (B, D) were measured in the 120 Li + 0 Ca2+ solution and are shown

as mean data ± S.E.M. before and after perfusion of MTSEA (A, B) and MTSET (C, D) reagents.

Experimental conditions were described earlier. Whole-cell current traces were measured under control

conditions (1-black line); after 30 s perfusion with 5 mM MTS (2-light gray line); after 5 min perfusion

(3-gray line); and after washout of the unreacted MTS (4-dark gray). MTSEA-induced covalent inhibition

of T528C and E535C was achieved within 30s since the averaged current traces recorded at 30 s were

superimposed to the current traces recorded after washout. MTSET- induced inhibition of E535C required

longer perfusion times suggesting that the rate of accessibility was decreased in that case. No inhibition

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31

was observed after 5 min exposure to 5 mM MTSES for either mutant (not shown). See Table I for

complete values.

Figure 8. Computer-predicted secondary structures generated by PROFILES-3D (INSIGHT II) using

KcsA (PDB 1BL8) as the template. Structure homology was performed for the 499-568 region of ECaC-

TRPV5 with only part of the actual S5 and S6 transmembrane regions. The domains were identified as

they appear in KcsA. The coiled structure following the selectivity filter is predicted to be longer in

ECaC-TRPV5 than in KcsA such as the α-helix TM2 (shown as a dotted box) starts at W87 in KcsA

whereas it starts at T558 in ECaC-TRPV5.

Figure 9. A. Ribbon 3-D representation of two ECaC-TRPV5 monomers obtained by homology

modeling using Modeller 6.2 as viewed from a perspective parallel to the membrane. The side-chains of

MTSET-modified positions in the pore helix region (T528, S532, E535, T539) and in the wild-type

channel at C556 are shown. D542 is projecting toward the selectivity filter. B. Surface 3-D representation

of ECaC-TRPV5 obtained by homology modeling using Modeller 6.2 as viewed from a perspective

parallel to the membrane. Residues are color-coded according to their reactivity toward MTSET.

Residues that were very significantly inhibited by MTSET at p < 0.001 are shown in red whereas

magenta-color residues were inhibited by MTSET at p < 0.01. Blue residues remained insensitive to

MTSET and non-functional residues are shown in yellow. White residues were not tested. D542 is shown

in green to indicate the region of the selectivity filter. The arrow indicates the direction of the ion fluxes.

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-150 -100 -50 50

-1,0

-0,8

-0,6

-0,4

-0,2

i / imax

Vm

MTSETMTSEA

-150 -100 -50 50

-1,0

-0,8

-0,6

-0,4

-0,2

i / imax

Vm

1- Ctrl2- 5 min3- Wash4- BMS

A. B.

C.1,4

3

2

1

2,3

4

-150 -100 -50 50

-1,0

-0,8

-0,6

-0,4

-0,2

i / imax

Vm -150 -100 -50 50

-1,0

-0,8

-0,6

-0,4

-0,2

i / imax

Vm

1

3

2

1

2,3

1- Ctrl2- 5 min3- Wash

D.

Figure 1

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Figure 2

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0 2 4 6 8 10 24 25-24

-21

-18

-15

-12

-9

-6

-3

0

min

-150 -100 -50 50

-1,0

-0,8

-0,6

-0,4

-0,2

i / imax

Vm

MTSETMTSEA

-150 -100 -50 50

-1,0

-0,8

-0,6

-0,4

-0,2

i / imax

Vm

A. B.

1- Ctrl2- 5 min3- Wash

1

3

2

1

2,3

C. D.

0 2 4 6 8 10 24 25

-10

-8

-6

-4

-2

0

min

MTSEA wash

i (µ

A)

i (µ

A)

MTSET wash

Figure 3

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-150 -100 -50 50

-1,0

-0,8

-0,6

-0,4

-0,2

i / imax

Vm -150 -100 -50 50

-1,0

-0,8

-0,6

-0,4

-0,2

i / imax

Vm

-150 -100 -50 50

-1,0

-0,8

-0,6

-0,4

-0,2

i / imax

Vm

MTSEA MTSET

MTSES

A. B.

C.

4

2,3

2,3,4

1- Ctrl2- 5 min3- Wash4- BMS

2,3

1

1

4

1

Figure 4

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Y526C

D525C

S524C

F523C

E522C

G521C

L520C

N519C

0 20 40 60 80 100% (i wash/i ctrl) at -150 mV

MTSES MTSET

S

5 lin

ker

-p

ore

: S

um

mar

y

**

**

**

*

Figure 5

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Pore helix Pore-S6 linkerA. B.

L538C

F537C

L536C

E535C

F534C

T533C

S532C

F531C

L530C

A529C

T528C

P527C

0 20 40 60 80 100 120% (i wash/i ctrl) at -150 mV

MTSES MTSET

****

*

C556

Y555C

M554C

F553C

P552C

L551C

D550C

V549C

S548C

Y547C

N546C

A545C

0 20 40 60 80 100% (i wash/i ctrl) at -150 mV

**

****

**

*

*

Figure 6

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MTSEA

T528C

MTSET

E535C-150 -100 -50 50

-1,0

-0,8

-0,6

-0,4

-0,2

i / imax

Vm

-150 -100 -50 50

-1,0

-0,8

-0,6

-0,4

-0,2

i/imax

Vm

-150 -100 -50 50

-1,0

-0,8

-0,6

-0,4

-0,2

i / imax

Vm

-150 -100 -50 50

-1,0

-0,8

-0,6

-0,4

-0,2

i / imax

Vm

A. B.

C. D.

1- Ctrl2- 30s3- 5 min4- Wash

2,3,4

1

2,3

4

1

24

1

3

2,4

1

3

Figure 7

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* KcsA: 35-LLVIVLLAGSYLAVLAERGAPGAQLITYPRALWWSVETATTVGYGDL-YPVTL-WGRCV--AVVVMVAGI-100 ECaC: 499-VVILGFASAFHITFQTEDPNNLGEFSDYPTALFSTFELFLTIIDGPANYSVDLPFMYCITYAAFAIIATL-568

Figure 8

Pore helix TM1 TM2

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D542

T539

T528

E535

S532

C556

Figure 9 A

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539

522

539 537

547

521521

540

522

Figure 9 B

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Simoes, Gérald Bernatchez, Rémy Sauvé and Lucie ParentYolaine Dodier, Umberto Banderali, Hélène Klein, Özlem Topalak, Omar Dafi, Manuel

Outer pore topology of the ECaC-TRPV5 channel by cysteine scan mutagenesis

published online November 20, 2003J. Biol. Chem.

10.1074/jbc.M310534200Access the most updated version of this article at doi:

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