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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Département de Physiologie and the Membrane Protein Research Group, Université de Montréal, Montréal, Québec, Canada
Submitted 5 September 2007 ; accepted in final form 11 October 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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calcium; kidney; transport; cysteine; site-directed mutagenesis; electrophysiology; methane thiosulfonate reagents; three-dimensional homology modeling; ion channel; transient receptor potential
Two extensive analyses of the outer vestibule of TRPV5 and TRPV6 channels have been published using a combination of substituted cysteine accessibility method (SCAM) and computer-based modeling (4, 23). The pattern of covalent modification by hydrophilic alkylthiosulfonate methane thiosulfonate (MTS) reagents in TRPV5 channels was found to support the predictions of a KcsA-based 3-D model, whereby the external vestibule encompassed a coiled structure called the turret connected to a small helical segment of 15 amino acids called the pore helix followed by the selectivity filter (4). Nonetheless, key questions regarding the structure of the selectivity filter could not be answered as the cysteine mutants at positions surrounding (and including) the high Ca2+ affinity Asp542 or Asp541 site were nonfunctional when expressed as homotetramers in TRPV5 (4) and TRPV6 (23) channels.
To examine the topology of the selectivity filter, the accessibility of nonfunctional residues was studied using tandems of covalently linked dimers. Based on the reactivity/accessibility to external membrane-impermeant MTS compounds of different charge and cross section, we conclude that four consecutive positions in the selectivity filter, including the high-affinity Ca2+-binding site Asp542, are easily accessible from the external aqueous medium. In addition, the side chain of Ile541, predicted to lie beneath Asp542, could be modified by MTS reagents provided that the side chain of Asp542 is substituted with a smaller and/or neutral residue. These results confirm that the side chains of the amino acids forming the selectivity filter are accessible from the external medium and that the selectivity filter of the Ca2+-selective TRPV5 channel is significantly wider than the selectivity filter of K+-selective channels, as measured from the atomic coordinates of their 3-D structures. More importantly, our data using I541C/D542G and I541C/D542N dimeric constructs indicate that the selectivity filter espouses a specific conformation in the presence of four carboxylate groups at position Asp542 such that removing two of the four carboxylate residues promotes the accessibility of Ile541 to MTS reagents.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Western blot analysis. Western blots were performed on total membranes from X. laevis oocytes obtained after two series of centrifugation, as detailed elsewhere (19). Proteins were analyzed on an 8% SDS-PAGE gel using primary antibody against the rabbit anti-rat TRPV5 (CaT2) channel from Alpha Diagnostic (1:100) and secondary horseradish peroxidase-conjugated anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:25,000). The quality of the overall procedure was monitored by Ponceau red staining.
Whole cell recordings. Whole cell currents were measured at room temperature with a two-electrode voltage-clamp amplifier (OC-725, Warner Instruments) as previously described (4, 9). Instantaneous current-voltage (I-V) 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. Under control conditions, I-V curves were recorded in the presence of nominally Ca2+-free Li+ solution containing (in mM) 120 LiOH, 5 EGTA, 2 KOH, and 20 HEPES titrated to pH 7.35 with methane sulfonic acid (9, 15). pCLAMP software (Clampex 8.1 and Clampex 9.2, Molecular Devices, Sunnyvale, CA) was used for online data acquisition and signal analysis. Unless stated otherwise, data were sampled at 10 kHz and low pass filtered at 5 kHz using the amplifier built-in filter. Data were analyzed using Origin 7.0 (OriginLab, Northampton, MA) software. Results are presented as means ± SE. An unpaired Students's t-test was used for statistical comparisons. Affinity for Ca2+ was assessed from the Ca2+ block of whole cell Li+ currents as previously described (4, 9, 16, 17). The stability constants used to calculate the free Ca2+ concentration were taken from Fabiato and Fabiato (5).
SCAM. The chemical structures of the MTS reagents used in this study are shown in Suppl Fig. 1.1 MTSEA and MTSET were purchased from Anatrace (Maumee, OH). 2-(Trimethyl ammonium)methyl methane thiosulfonate bromide (MTSMT), 2-(trimethyl ammonium)hexyl methane thiosulfonate bromide (MTSHT), and MTSES were purchased from Toronto Research Chemicals (Toronto, ON, Canada). Because MTS reagents are rapidly hydrolyzed (5–10 min), they were always prepared fresh before use, as previously described (4, 22). Whole cell current traces were recorded in Ca2+-free Li+ solution (see Whole cell currents) using voltage ramps imposed every 30 s up to 5 min after exposure to MTS reagents (4). Averaged whole cell current traces are shown for control conditions, 30 s after bath addition of the MTS reagent, at 2 min, at 5 min, and after washout of the unreacted MTS reagent for 10 min with the control solution. In addition to providing the MTS response over a wide range of potentials, voltage ramps allowed us to check for the presence of rectifying currents throughout the course of an experiment. For most mutants, the gradual increase in nonspecific currents or leaky currents could be immediately assessed from the gradual disappearance of the trademark inward rectification of TRPV5 currents. Experiments where nonspecific leaks developed during the experiment were simply discarded. The percentage of inhibition of whole cell currents remaining after a 5-min exposure to MTS reagent was computed at a membrane potential of –150 mV after MTS washout compared with whole cell currents measured under control conditions before MTS application. Inhibition was deemed "significant" at P < 0.01.
Computer-predicted structure and homology modeling of the TRPV5 channel.
We used the SAMT06 server to identify structural templates for the external vestibule of the TRPV5 channel. The SAMT06 server, which includes primary sequence alignment coupled with secondary structure fold alignment in its scoring function, proposed Kv1.2 (2A79.pdb) as a structural template for the external vestibule of the TRPV5 channel between S5 and the end of S6. Both the KcsA-based (4) and Kv1.2- based 3-D models agree with the same structural domains, although they differ slightly in their boundaries. Homology models based on the atomic coordinates of KcsA models are believed to represent the structural features of a channel in the closed state (11). To more accurately represent the structural features of the TRPV5 channel in the open state (since SCAM data are being gathered in the channel open state), we built a symmetrical 3-D model of the channel using the molecular coordinates of the tetrameric Kv1.2 channel (2A79.pdb1). Unlike the KcsA-based model (4), the Kv1.2-based homology model predicts a wide inner vestibule with a bend at Leu573 in S6. The resulting outer vestibule encompasses three structural domains consisting of a coiled structure (Glu515 to Tyr526) connected to a small helical segment of 13 amino acids (527-PTALFSTFELFLT-539) followed by a distinct coiled structure from Ile541 to Asn546 (selectivity filter) that spreads until the beginning of S6 at Pro552. According to the alignment provided by SAMT06, the signature sequence GYGD in K+ channels is aligned without any gap with the IDGP sequence of the TRPV5 channel (Fig. 2). The selectivity filter of the Kv1.2-based 3-D model includes Ile541, suggesting that its side chain could be accessible from the extracellular medium. The Kv1.2-based model shows that the Ala545 to Cys556 linker region forms a coiled structure considerably longer than predicted from the KcsA-based model (4). Modeling was carried out with Modeller version 9.1 (20). Spatial constraints were included to accommodate the four carboxylate groups of Asp542 and minimize interaction energy within the selectivity filter. Interatomic distances within these carboxylate groups were based on the crystal coordinates of calmodulin using the average distance between Ca2+ and carboxylate groups of key aspartate residues (2F3Y.pdb). This results in a selectivity filter with a diameter of 8–10 Å (C
to C), which is significantly larger than the diameter of 3–4 Å estimated from the 3-D structure of crystallized K+ channels. Because of the poor sequence homology between Kv1.2 and TRPV5 channels in coiled regions, the two extracellular linkers (the S5 to pore helix linker called the turret and the selectivity filter to S6 linker) were modeled de novo using parameters provided by Modeller version 9.1 (20). Automated homology modeling involved the generation of 150 models (see 
Fig. 8). The model with the lowest objective function and the lowest root mean square deviation compared with the template was retained, and residues facing the external medium were minimized with the force fields included within the DISCOVER module of INSIGHTII using a dielectric constant of 80 to take into account the presence of solvent. The structural quality of the final model was evaluated by PROCHECK and ProQ. The 3-D representation of the TRPV5 channel was generated with INSIGHTII software (Accelrys, San Diego, CA), as described elsewhere (4, 22). The atomic coordinates of the 3-D Kv1.2 homology-based model of the TRPV5 channel are provided at the end of the Supplemental Data.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The control C556S dimeric construct (C556S_C556S) showed little inhibition by either MTS reagent including MTSEA, which is considered to be potentially membrane permeant (8) (Figs. 3A and 4 and Suppl. Table 1). In contrast, 75 ± 7% (n = 4) of the D542C dimer whole cell currents were inhibited by 1 mM MTSET within 30 s of exposure. No further inhibition was observed after 5 min of perfusion (Fig. 3B). Similar inhibition of the D542C dimer by MTSEA and MTSMT demonstrated that the methyl chain of MTSMT is long enough to reach position Asp542 and that the small NH3 head group of MTSEA is sufficient to avert cation fluxes (Fig. 4). Depending on the distance of Asp542 from the external medium, it could also suggest that the pore is wide enough to accommodate the 5.8-Å head of MTSET. The inhibition by MTSES was comparable with the ones reported for positively charged MTS reagents, suggesting that the two remaining aspartine residues at position 542 in the tetrameric channel did not repel the negatively charged head group of MTSES. In addition, functional expression of the D542C dimer significantly decreased Ca2+ affinity from an IC50 of 2.2 ± 0.4 µM (n = 9) (9) for the wild-type channel to an IC50 of 150 ± 6 µM (n = 5) in addition to a lessening of inward rectification, as expected since Asp542 is the key high-affinity Ca2+-binding site as well as one of the key sites for the Mg2+-induced voltage-dependent block that is responsible for channel inward rectification (10, 14). The decrease in Ca2+ affinity was roughly 5- to 10-fold lower than reported elsewhere for the tetrameric D542N mutation (9), suggesting that the four carboxylate groups found at position 542 in the symmetrical tetrameric channel work in an additive fashion to form the high-affinity Ca2+-binding site. Permeation properties of D542C were also altered. The mean open time increased significantly, and the single channel conductance of the D542C dimer was significantly reduced from 46 ± 3 pS (n = 5) for the control dimer to 28 ± 1 pS (n = 2; Suppl Fig. 2). The latter probably accounts for the observation that whole cell currents generated by the D542C dimer remained significantly smaller than for most TRPV5 mutations (Suppl. Table 1).
Residues in the selectivity filter are easily accessible to positively charged MTS reagents. As already shown elsewhere, most cysteine mutations in the selectivity filter failed to express whole cell currents when expressed as homotetramers (4). Information regarding the accessibility of the side chains at these positions was thus lacking. Accessibility of the residues lining the selectivity filter (I541C to Y547C) was thus analyzed using dimeric constructs (4). One of the key biophysical features of the control C556S subunit, namely, the steep inward rectification, was preserved in all the dimeric constructs but for the D542C construct, suggesting that the selectivity filter was not grossly distorted by the cysteine mutation. In addition, 30–50% of whole cell Li+ currents generated by cysteine dimeric constructs were inhibited by 1 µM free Ca2+, a value that is similar to the wild-type channel (9). Significant inhibition of dimers was observed at four consecutive positions from 542 to 545 and at position 547 after exposures to positively charged MTS reagents (MTSET, MTSEA, and MTSMT; Figs. 4 and 5, Suppl. Fig. 3, and Suppl. Table 1). Figure 5 shows average I-V curves measured at 30 s, 2 min, 5 min, and after MTSET washout. Maximal inhibition was achieved within 30 s of exposure to MTSET (Fig. 5) for G543C, P544C, and A545C. Incubation periods as long as 5 min did not increase the steady-state inhibition for either construct. Inhibition by shorter chain MTSMT was at once rapid (<30 s) and complete for all these constructs. MTSEA inhibition was generally slower for all constructs but P544C, reaching a maximum after a 2-min exposure and failing to increase after a 5-min incubation period (Suppl. Fig. 3). Longer chain MTSHT, with a hexyl chain spanning a distance of 14.5 Å compared with 8.9 Å for MTSET (3), produced a similar inhibition of the four consecutive dimeric constructs from D542C to A545C (Suppl. Fig. 4). Altogether, these data suggest that the volume of the head group [N(CH3)3 vs. NH3] was more important for steady-state inhibition than the length of the alkly chain (ethyl vs. methyl). Positions 542, 543, 544, and 547 were seen to be inhibited to the same extent by all positively charged MTS reagents, whereas inhibition of A545C was significantly (P < 0.001) stronger with MTSMT, which bears a methyl chain that is shorter than the ethyl chain of MTSET. This result suggests a superficial location for the cysteine-substituted 545 position such that the trimethyl amine head would be more effective to inhibit ion fluxes when tethered on a chain shorter by 1–2 Å. Altogether, these results indicate that the four contiguous positions in the selectivity filter of the TRPV5 channel are accessible to positively charged MTS reagents from the external medium. No inhibition was observed with I541C and N546C tandem constructs with either positively or negatively charged MTS reagents (Fig. 4). The absence of modification at position 546 indicates that 546C is either not accessible from the aqueous medium or that covalent modification by MTS reagents does not impede ion permeation through the TRPV5 channel, unlike its neighbors 545C and 547C.
Reactivity to negatively charged MTSES is residue dependent. Significant inhibition by negatively charged MTSES was obtained at positions D542C, P544C, and A545C but not at positions 543 and 547 (Fig. 4, Suppl. Fig. 5, and Suppl. Table 1). The reasons for the absence of inhibition by MTSES at these positions are not known. Nonetheless, given that MTSES reactivity was absent in the entire pore helix region (P527-T539) (4), the inhibition of some cysteine mutants by MTSES in the selectivity filter confirms that the selectivity filter behaves like a wide open coiled structure.
Investigation of the role of carboxylate side chains in controlling MTS access to the selectivity filter. Although presumed to be part of the selectivity filter, the I541C dimeric construct showed no significant inhibition by either positively or negatively charged MTS reagents (Fig. 4). Three possible interpretations can account for this result: 1) Ile541 is located too deep within the filter to be accessible from the external medium with the ethyl chain of MTSET; 2) Ile541 faces the intracellular cavity; or 3) the presence of the large aspartate residue (111 Å3) nearby (Asp542) blunts access to MTS reagents. It can indeed be argued that the presence of four charged carboxylate residues in the native channel at position 542 hinders access of MTS reagents to I541C. MTS reactivity of dimeric I541C was thus investigated in the I541C/D542G dimeric construct, where both I541C and D542G are produced on the same subunit. D542G has already been shown to express large inward Li+ currents (9). As shown in Figs. 6 and 7, the I541C/D542G dimeric construct was significantly inhibited by MTSEA, MTSMT, MTSET, and MTSES. Hence, the side chain at position I541 is potentially accessible from the external medium providing that the carboxylate side chains of Asp542 do not hinder MTS accessibility. The presence of the smaller and neutral glycine (48 Å3) in the selectivity filter at two of the four positions occupied by aspartate residues in the wild-type channel could improve accessibility by lessening steric hindrance, decreasing the negative electrostatic charge in the filter, or by reducing both. In regard to electrostatics, the net negative charge of the selectivity filter could improve access to positively charged MTS reagents, whereas it could repel negatively charged MTSES. Electrostatic effects were addressed by studying the MTS inhibition of dimeric constructs D542G/G543C and D542G/P544C. If negatively charged carboxylate groups modify the electrostatic environment, it is expected that the inhibition by positively charged MTS reagents will decrease, whereas the inhibition by negatively charged MTSES could be enhanced, by the D542G mutation. As can be seen by comparing Figs. 4 and 7, replacement of two of the four carboxylate residues by hydrogen atoms at position 542 did not significantly affect the steady-state inhibition by either MTS reagent, although the rate of modification was altered. In particular, introduction of the neutral glycine residue at position 542 failed to improve the steady-state inhibition of G543C by negatively charged MTSES, ruling out a significant contribution of Asp542 in the MTSES reactivity of side chain residues in the upper section of the selectivity filter. Nonetheless, these data confirm that G543C and P544C are closer to the extracellular medium than Asp542, as predicted by KcsA- and Kv1.2-based 3-D models.
To investigate the role of steric hindrance in the accessibility of I541C, the MTS reactivity of the I541C/D542N dimeric construct was evaluated (Fig. 6). In this construct, the aspartate (111 Å3) at position 542 was replaced with neutral but polar asparagine (114 Å3). Figure 7 shows that accessibility to positively charged MTSET and MTSMT reagents was comparable with the one measured in the I541C/D542G construct, suggesting steric hindrance alone cannot explain the complete lack of reactivity of the simple I541C dimeric construct. Nonetheless, steady-state inhibitions of I541C/D542N channels by MTSEA and MTSES were less important than that observed with the I541C/D542G dimeric construct, suggesting that the structure of the selectivity filter was somewhat different in the presence of the D542N versus D542G mutation. We (4) have previously shown I540C to be insensitive to functional modification by MTS reagents, but we were unable to determine if this resulted from the presence of Asp542 as the I540C/D542G channel was nonfunctional as homotetramers (Suppl. Table 1).
Structural features of the external linkers in the TRPV5 channel. Our previous work has already established the relative accessibility of residues at positions 548, 549, and 550 within the linker connecting the selectivity filter to S6, but cysteine mutants 551C and 552C were not functional as homotetramers (4). When assessed using tandems of dimers, 551C and 552C were strongly inhibited by positively charged MTS reagents (Suppl. Fig. 6 and Suppl. Table 2). This result suggests that this region predicted to form a coiled structure is not only accessible from the extracellular medium but that residues at positions 551 and 552 could be located close enough to the permeation pathway to inhibit monovalent cation fluxes. Save for position 551, inhibition by negatively charged MTSES remained near absent in the region. These data contrast with the absence of reactivity from the N546C dimeric construct, which is predicted from the 3-D model to be closer to the permeation pathway than both positions 551 and 552.
The strongest and fastest inhibitions of cysteine-substituted side chains in the outer pore were nonetheless reported with the homotetrameric E522C channel (4) located in the S5 to pore helix linker, a region that is predicted to lie away from the permeation pathway (Suppl. Fig. 7). To evaluate whether the extent of inhibition was controlled by the number of cysteine residues present in the outer pore, we measured the MTS reactivity of E522C dimers. Strong inhibitions were observed with both positively and negatively charged MTS reagents (Suppl. Fig. 8). Taken together, these data highlight the limitations of using computer-based homology models to picture the 3-D structure of coiled regions such as extracellular linkers.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The side chains of four consecutive positions in the selectivity filter from Asp542 to Ala545 and Tyr547 were shown to be easily accessible from the external medium. In addition, the Ile541 position became accessible when two of the four carboxylate residues at position 542 were replaced by either a smaller glycine residue or a similarly sized but neutral aspartate residue. The inhibition of the I541C/D542N dimeric construct by positively and negatively charged MTS reagents ruled out steric hindrance as the sole determinant of accessibility within the selectivity filter. In particular, the rapid modification of the I541C/D542N dimeric construct by negatively charged MTSES suggests that the residue at position 541 is not much influenced by the local electrical field. It could be speculated that the selectivity filter espouses a specific conformation in the presence of four carboxylate residues such that disrupting the ring with two neutral aspartate residues promotes the accessibility of Ile541 to MTS reagents. This interpretation rests on the assumption, upon which rests any SCAM analysis, that the cysteine substitution does not alter the orientation of the side chain. Together with the 3-D model of the TRPV5 channel in the open state, our data further suggest that the side chains of Ile541 and Asp542 could be potentially accessible both from the extracellular and intracellular medium, a conclusion that could simply not be reached from the tepee-shaped KcsA-based models of TRPV5 (4) and TRPV6 (23) channels. It is already known that inward rectification is caused in part by the binding of internal Mg2+ to the aspartate residue within the selectivity filter (24). It thus remains to be seen whether the bulky side chain of isoleucine could itself influence the diffusion of internal divalent cations that are believed to reach Asp542 from the intracellular medium and block outward currents in a voltage-dependent manner (24).
According to the Kv1.2-based model of the selectivity filter (Fig. 8), Asp542 would be equivalent to the S1 high-affinity K+-binding site in the high-resolution KcsA structure (26) and subsequent 543, 544, and 545 positions would be located in the extracellular linker that follows the selectivity filter and precedes S6. A superficial location for the cysteine-substituted 545 residue poised at the extracellular mouth of the channel, as shown in our current Kv1.2-based model, could account for the stronger inhibition observed at position 545 with short-chain MTSMT compared with MTSET. The equivalent position in the TRPV6 channel (A544C) was also shown to be significantly modified by positively charged MTSEA and MTSET. In addition, we demonstrated that negatively charged MTSES could modify side chains at positions 542, 544, and 545, indicating that the local electrical field does not repulse negatively charged reagents in this region. These observations suggest the presence of large number of water molecules dampening down the effect of the electrical field in this region and could be explained if these residues are facing and/or bathing in the external medium, as suggested by the 3-D model.
Remarkably, cation fluxes through N546C remained unaffected by the covalent modification by MTS reagents. Our data suggest that this residue adopts, in average, a position that is not facing the permeation pathway such that modification by any MTS reagents remained silent and failed to modify ion fluxes. The resolution of our 3-D model in the linker regions is too low to represent a valid picture of the channel conformation. Even with high-resolution X-ray data, the positions of the atoms as well as the projection of the side chains in the coiled linkers remain less precise than in helicodial regions. These limitations of the 3-D model are also illustrated with the SCAM data of the L551C and E522C dimeric constructs. Both positions are predicted to lie away from the permeation pathway, yet both positively and negatively charged MTS reagents produced fast and complete inhibition of cation fluxes when covalently linked to any of these positions. Taken together, these SCAM data are a sharp reminder that 3-D models must be validated with functional data.
Although the TRPV5 channel is an atypical TRP channel, the conclusions from our work could be safely extended to the related TRPV6 channel. There are only two substitutions in the primary sequence of the selectivity filter, with a serine to aspartate substitution at position 548 and a cysteine to serine change at position 556 from the TRPV5 to TRPV6 channel. Position 548 was studied elsewhere in a purely symmetrical tetrameric channel and was shown to remain insensitive to MTS inhibition in TRPV5 (4) and TRPV6 (23) channels. The second substitution was eliminated in the context of our SCAM experiments, as C556S was the template for all our constructs. Hence, although long speculated, this is the first study to clearly demonstrate that the side chains of the aspartate residue key for forming the high-affinity binding site for Ca2+ in TRPV5 or TRPV6 channels are accessible to MTS reagents from the extracellular medium. While the selectivity filter of the related TRPV6 channel has been previously studied by SCAM (23), D541C in the TRPV6 channel remained nonfunctional when tested as a rescued subunit.
Our SCAM data on the Ile541 to Tyr547 region could suggest that the selectivity filter of the TRPV5 channel is much wider than the selectivity filter of K+ channels, similar to the nonselective cationic NaK channel (21). It could also suggest that this whole region is closer to the extracellular medium and shorter than the selectivity filter of K+ channels, with Asp542 being equivalent to the S0 K+-binding site instead of the S1 site. Such a model could be obtained by introducing gaps in the primary sequence alignment such that Asp542 would be aligned with the underlined valine in the GYGDVV sequence. This question may form the basis for future structural studies of TRPV5/6 channels.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supplemental material for this article is available online at the American Journal of Physiology-Cell Physiology website. 
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