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• W2023463047 abstract "The TRP superfamily forms a functionally important class of cation channels related to the product of the Drosophila trp gene. TRP channels display an unusual diversity in activation mechanisms and permeation properties, but the basis of this diversity is unknown, as the structure of these channels has not been studied in detail. To obtain insight in the pore architecture of TRPV6, a Ca2+-selective member of the TRPV subfamily, we probed the dimensions of its pore and determined pore-lining segments using cysteine-scanning mutagenesis. Based on the permeability of the channel to organic cations, we estimated a pore diameter of 5.4 Å. Mutating Asp541, a residue involved in high affinity Ca2+ binding, altered the apparent pore diameter, indicating that this residue lines the narrowest part of the pore. Cysteines introduced in a region preceding Asp541 displayed a cyclic pattern of reactivity to Ag+ and cationic methylthio-sulfanate reagents, indicative of a pore helix. The anionic methanethiosulfonate ethylsulfonate showed only limited reactivity in this region, consistent with the presence of a cation-selective filter at the outer part of the pore helix. Based on these data and on homology with the bacterial KcsA channel, we present the first structural model of a TRP channel pore. We conclude that main structural features of the outer pore, namely a selectivity filter preceded by a pore helix, are conserved between K+ channels and TRPV6. However, the selectivity filter of TRPV6 is wider than that of K+ channels and lined by amino acid side chains rather than main chain carbonyls. The TRP superfamily forms a functionally important class of cation channels related to the product of the Drosophila trp gene. TRP channels display an unusual diversity in activation mechanisms and permeation properties, but the basis of this diversity is unknown, as the structure of these channels has not been studied in detail. To obtain insight in the pore architecture of TRPV6, a Ca2+-selective member of the TRPV subfamily, we probed the dimensions of its pore and determined pore-lining segments using cysteine-scanning mutagenesis. Based on the permeability of the channel to organic cations, we estimated a pore diameter of 5.4 Å. Mutating Asp541, a residue involved in high affinity Ca2+ binding, altered the apparent pore diameter, indicating that this residue lines the narrowest part of the pore. Cysteines introduced in a region preceding Asp541 displayed a cyclic pattern of reactivity to Ag+ and cationic methylthio-sulfanate reagents, indicative of a pore helix. The anionic methanethiosulfonate ethylsulfonate showed only limited reactivity in this region, consistent with the presence of a cation-selective filter at the outer part of the pore helix. Based on these data and on homology with the bacterial KcsA channel, we present the first structural model of a TRP channel pore. We conclude that main structural features of the outer pore, namely a selectivity filter preceded by a pore helix, are conserved between K+ channels and TRPV6. However, the selectivity filter of TRPV6 is wider than that of K+ channels and lined by amino acid side chains rather than main chain carbonyls. The TRP superfamily consists of cation channels related to the product of the Drosophila trp (for transient receptor potential) gene (1Nilius B. Cell Calcium. 2003; 33: 293-298Crossref PubMed Scopus (69) Google Scholar, 2Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (947) Google Scholar). Based on sequence homology, the 21 mammalian TRPs can be subdivided into three subfamilies (3Harteneck C. Plant T.D. Schultz G. Trends Neurosci. 2000; 23: 159-166Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 4Montell C. Birnbaumer L. Flockerzi V. Bindels R.J. Bruford E.A. Caterina M.J. Clapham D.E. Harteneck C. Heller S. Julius D. Kojima I. Mori Y. Penner R. Prawitt D. Scharenberg A.M. Schultz G. Shimizu N. Zhu M.X. Mol. Cell. 2002; 9: 229-231Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar) as follows: members of TRPC subfamily (the C stands for canonical) display the highest homology to Drosophila TRP, members of the TRPV subfamily are most homologous to the vanilloid receptor 1 (VR1, now TRPV1), and members of TRPM subfamily show the highest homology with melastatin (TRPM1). TRP channels are implicated in a variety of physiological processes, ranging from phospholipase C- and/or store-dependent Ca2+ influx in most cell types to reabsorption of Ca2+ and Mg2+ in kidney and intestine, and detection of chemical and thermal signals in the sensory system (1Nilius B. Cell Calcium. 2003; 33: 293-298Crossref PubMed Scopus (69) Google Scholar, 2Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (947) Google Scholar, 5Voets T. Nilius B. J. Membr. Biol. 2003; 192: 1-8Crossref PubMed Scopus (67) Google Scholar, 6Montell C. Science's STKE. 2001; http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2001/2090/re2001PubMed Google Scholar). This functional diversity is reflected in a wide variety of biophysical properties and gating mechanisms among the different TRP family members and subfamilies.Like voltage-gated K+ channels and cyclic nucleotide-gated channels, functional TRP channels are homo- and/or heteromers of four subunits, each containing six transmembrane (TM) 1The abbreviations used are: TM, transmembrane; MTS, methanethiosulfonate; MTSEA, methanethiosulfonate ethylammonium; MTSES, methanethiosulfonate ethylsulfonate; MTSET, [2-(trimethylammonium) ethyl]methanethiosulfonate bromide; WT, wild type; MA+, methylammonium; DMA+, dimethylammonium; NMDG+, N-methyl-d-glucamine; SCAM, substituted cysteine accessibility method; DVF, divalent cation-free; TriMA+, trimethylammonium; TetMA+, tetramethylammonium. 1The abbreviations used are: TM, transmembrane; MTS, methanethiosulfonate; MTSEA, methanethiosulfonate ethylammonium; MTSES, methanethiosulfonate ethylsulfonate; MTSET, [2-(trimethylammonium) ethyl]methanethiosulfonate bromide; WT, wild type; MA+, methylammonium; DMA+, dimethylammonium; NMDG+, N-methyl-d-glucamine; SCAM, substituted cysteine accessibility method; DVF, divalent cation-free; TriMA+, trimethylammonium; TetMA+, tetramethylammonium. domains, cytoplasmic N and C termini, and a putative pore region between TM5 and TM6 (7Hoenderop J.G. Voets T. Hoefs S. Weidema F. Prenen J. Nilius B. Bindels R.J. EMBO J. 2003; 22: 776-785Crossref PubMed Scopus (293) Google Scholar). However, in comparison with most other ion channel families, little is known about the structure of TRP channels, and the structural organization of their cation-selective pores is still poorly understood (8Voets T. Nilius B. Cell Calcium. 2003; 33: 299-302Crossref PubMed Scopus (35) Google Scholar). There is a striking variability in the permeation properties within the TRP superfamily, which includes channels that are rather non-selective for mono- and divalent cations (e.g. TRPV1) (9Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (6943) Google Scholar), permeable for monovalent cations but impermeable for Ca2+ (e.g. TRPM4 and TRPM5) (10Launay P. Fleig A. Perraud A.L. Scharenberg A.M. Penner R. Kinet J.P. Cell. 2002; 109: 397-407Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 11Hofmann T. Chubanov V. Gudermann T. Montell C. Curr. Biol. 2003; 13: 1153-1158Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar), or highly selective for Ca2+ over monovalent cations (TRPV5 and TRPV60 (12Vennekens R. Hoenderop J.G. Prenen J. Stuiver M. Willems P.H. Droogmans G. Nilius B. Bindels R.J. J. Biol. Chem. 2000; 275: 3963-3969Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 13Yue L. Peng J.B. Hediger M.A. Clapham D.E. Nature. 2001; 410: 705-709Crossref PubMed Scopus (316) Google Scholar, 14Hoenderop J.G. Vennekens R. Müller D. Prenen J. Droogmans G. Bindels R.J. Nilius B. J. Physiol. (Lond.). 2001; 537: 747-761Crossref Scopus (225) Google Scholar). This variability contrasts with most other families of ion channels, where the differences in permeation properties within one family are generally small (15Hille B. Ion Channels of Excitable Membranes. 3rd Ed. Sinauer Associates, Inc., Sunderland, MA2001Google Scholar). At present, the origin of these large differences in pore properties within the TRP superfamily is fully unknown.The present study provides the first systematic analysis of the pore region of a member of the TRP superfamily. We focused on TRPV6, a channel that has been implicated in Ca2+ reabsorption in the intestine and kidney (16Peng J.B. Brown E.M. Hediger M.A. J. Physiol. (Lond.). 2003; 551: 729-740Crossref Scopus (104) Google Scholar), which is an especially interesting model channel for two reasons. First, together with the highly homologous TRPV5, it displays by far the highest Ca2+ selectivity reported for a TRP channel (12Vennekens R. Hoenderop J.G. Prenen J. Stuiver M. Willems P.H. Droogmans G. Nilius B. Bindels R.J. J. Biol. Chem. 2000; 275: 3963-3969Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 13Yue L. Peng J.B. Hediger M.A. Clapham D.E. Nature. 2001; 410: 705-709Crossref PubMed Scopus (316) Google Scholar, 14Hoenderop J.G. Vennekens R. Müller D. Prenen J. Droogmans G. Bindels R.J. Nilius B. J. Physiol. (Lond.). 2001; 537: 747-761Crossref Scopus (225) Google Scholar). The permeability ratio for Ca2+ over Na+ of >100 reported for TRPV5 and TRPV6 is comparable only to that of voltage-gated Ca2+ channels and the calcium release-activated calcium channel (CRAC) (15Hille B. Ion Channels of Excitable Membranes. 3rd Ed. Sinauer Associates, Inc., Sunderland, MA2001Google Scholar, 17Hoth M. Penner R. Nature. 1992; 355: 353-356Crossref PubMed Scopus (1482) Google Scholar). Second, a stretch of 20 amino acids between TM5 and TM6 of TRPV6 displays ∼40% sequence identity with a region in the bacterial K+ channel KcsA that, based on the KcsA crystal structure (18Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5688) Google Scholar), forms the pore helix and selectivity filter. Such a high degree of sequence identity with K+-selective pores is only seen for members of the TRPV subfamily (8Voets T. Nilius B. Cell Calcium. 2003; 33: 299-302Crossref PubMed Scopus (35) Google Scholar) and might point toward similarities in the structure of TRP and K+ channels. Two distinct approaches were used to obtain insight in the TRPV6 pore. First, we measured the permeability of the channel to cations of increasing size to estimate of the diameter of the channel pore at its narrowest point, and we identified mutants with altered pore diameter. Second, we made use of the substituted cysteine accessibility method (SCAM) (19Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (542) Google Scholar) to identify pore-lining residues. Based on our findings, we propose a structural model that encompasses the position of residues determining the cation, size, and Ca2+ selectivity of the TRPV6 pore.EXPERIMENTAL PROCEDURESCell Culture, Transfection, and Mutagenesis—HEK-293 cells were grown in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal calf serum, 2 mm l-glutamine, 2 units/ml penicillin, and 2 mg/ml streptomycin at 37 °C in a humidity-controlled incubator with 10% CO2. They were transiently transfected with the pCINeo/IRES-GFP/mCaT1 vector (14Hoenderop J.G. Vennekens R. Müller D. Prenen J. Droogmans G. Bindels R.J. Nilius B. J. Physiol. (Lond.). 2001; 537: 747-761Crossref Scopus (225) Google Scholar) using methods described previously (20Trouet D. Nilius B. Voets T. Droogmans G. Eggermont J. Pfluegers Arch. 1997; 434: 632-638Crossref PubMed Scopus (62) Google Scholar), and electrophysiological recordings were performed between 8 and 24 h after transfection. Single amino acids in the pore region of TRPV6 were mutated to cysteines using the standard PCR overlap extension technique (21Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6797) Google Scholar), and the nucleotide sequences of all mutants were verified by sequencing of the corresponding cDNAs.Coexpression of mutant and WT TRPV6 was obtained by mixing the corresponding vector DNAs in a 3 to 1 ratio. Assuming a random assembly of WT and mutant subunits, we predict that >99% of the tetrameric channels that are formed under these conditions contain at least one mutant subunit (7Hoenderop J.G. Voets T. Hoefs S. Weidema F. Prenen J. Nilius B. Bindels R.J. EMBO J. 2003; 22: 776-785Crossref PubMed Scopus (293) Google Scholar). As under this condition no functional expression could be obtained for mutants L537C, D541C, and Y546C, we also tried other ratios of WT and mutant vector DNAs (1 to 1 and 1 to 3). However, this also did not yield any evidence for the incorporation of these three mutant subunits into a functional channel.Electrophysiology—Transfected cells were identified by their green fluorescence when illuminated at 480 nm using the polychrome IV monochromator (T.I.L.L. Photonics, GmbH). Patch clamp experiments were performed in the tight seal whole-cell configuration at room temperature (20–25 °C), using an EPC-9 patch clamp amplifier and Pulse software (HEKA Electronics). Patch pipettes had DC resistances of 2–4 megohms when filled with intracellular solution. Series resistances were between 3 and 10 megohms and were compensated 60–80%. Residual voltage errors due to uncompensated series resistances were generally <10 mV, and cells with larger voltage errors were omitted from the analysis. All potentials were corrected for possible liquid junction potentials, which were calculated according to Barry (22Barry P.H. J. Neurosci. Methods. 1994; 51: 107-116Crossref PubMed Scopus (545) Google Scholar). A 200-ms voltage ramp ranging from -150 to +100 mV was applied at a frequency of 0.5 Hz from a holding potential of 0 mV. Currents were sampled at 10 kHz and filtered at 2.9 kHz using an eight-pole Bessel filter. The time course of the whole-cell current was obtained by averaging the inward current in a narrow voltage window around -80 mV. Current amplitudes in divalent cation-free solution (see below) measured in cells expressing WT TRPV6 or the different functional mutants used in this study were on average approximately 2 orders of magnitude larger (between 3 and 30 nA at -80 mV) than the background current in non-transfected cells (-73 ± 8pAat -80 mV; n = 9). For that reason, background currents were not taken into account during analysis.Solutions—The standard divalent cation-free (DVF) extracellular solution contained (in mm): 150 NaCl, 10 EDTA and 10 Hepes, titrated to pH 7.4 with NaOH. The permeability of ammonium and its mono-, di-, tri-, and tetramethyl substituents was tested using DVF solutions in which all Na+ was substituted by the respective cations. In these permeation experiments, the standard DVF solution was used as intracellular solution. To test the accessibility of substituted cysteines, MTSEA, MTSET, or MTSES (Toronto Research Chemicals) was added to the standard DVF solution from 100 mm stocks, which were prepared daily and kept on ice until usage. Because of the low solubility of AgCl, modification by Ag+ was tested in a modified, Cl--free DVF solution, which contained 150 mm NaNO3 instead of NaCl. AgNO3 was added to this solution at concentrations of 1 or 10 μm, which yields free Ag+ concentrations of 20 or 200 nm, respectively, taking into account the chelating action of EDTA. The intracellular solution for the cysteine modification experiments contained (in mm): 150 NaCl, 3 MgCl2, 10 EGTA and 10 Hepes, titrated to pH 7.4 with NaOH. When indicated, this solution was supplemented with 10 mm cysteine.Data Analysis—Data analysis and display were done by using Microcal Origin software version 7.0 (OriginLab Corporation). Data are shown as mean ± S.E. from at least four cells.RESULTSDetermination of the Pore Diameter of WT TRPV6 and Location of the Narrowest Part of the Pore—To estimate the diameter of the TRPV6 permeation pore, we measured the relative permeability of organic monovalent cations of increasing size. These experiments were performed with EDTA-buffered divalent-free intra- and extracellular solutions to avoid the potent blocking of monovalent currents by Mg2+ and Ca2+ (14Hoenderop J.G. Vennekens R. Müller D. Prenen J. Droogmans G. Bindels R.J. Nilius B. J. Physiol. (Lond.). 2001; 537: 747-761Crossref Scopus (225) Google Scholar, 23Voets T. Prenen J. Fleig A. Vennekens R. Watanabe H. Hoenderop J.G. Bindels R.J. Droogmans G. Penner R. Nilius B. J. Biol. Chem. 2001; 276: 47767-47770Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 24Voets T. Janssens A. Prenen J. Droogmans G. Nilius B. J. Gen. Physiol. 2003; 121: 245-260Crossref PubMed Scopus (136) Google Scholar), which would complicate accurate measurement of the reversal potential. With Na+ as the sole intra- and extracellular cation, TRPV6 currents reversed close to 0 mV and showed significant inward rectification, which has been shown previously to be an intrinsic property of the pore (24Voets T. Janssens A. Prenen J. Droogmans G. Nilius B. J. Gen. Physiol. 2003; 121: 245-260Crossref PubMed Scopus (136) Google Scholar). Subsequently, all Na+ in the extracellular solution was replaced by ammonium or its mono-, di-, tri-, or tetramethyl substituents (Fig. 1A). Clear inward currents were recorded with ammonium (A+; diameter (a) = 3.4 Å), monomethylammonium (MA+; a = 3.6 Å), and dimethylammonium (DMA+; a = 4.6 Å). We determined permeability ratios relative to Na+ (PX/PNa) from the biionic reversal potentials (15Hille B. Ion Channels of Excitable Membranes. 3rd Ed. Sinauer Associates, Inc., Sunderland, MA2001Google Scholar), which yielded values of 1.63 ± 0.07, 0.49 ± 0.03, and 0.09 ± 0.01 for A+, MA+, and DMA+, respectively. Trimethylammonium (TriMA+; a = 5.2 Å) and tetramethylammonium (TetMA+; a = 5.8 Å) failed to carry significant inward current and shifted the reversal potential to voltages more negative than -120 mV, indicating that the permeability of the channel to these cations is extremely low or zero (PX/PNa < 0.01). A similar negative reversal potential was obtained with the even larger N-methyl-d-glucamine (NMDG+; a = 6.8 Å) as the sole extracellular cation.It has been shown previously that the high Ca2+ and Mg2+ sensitivity of TRPV5 and TRPV6 is determined by a single aspartate residue (Asp542 in TRPV5 and Asp541 in TRPV6) in the pore region between TM5 and TM6 (24Voets T. Janssens A. Prenen J. Droogmans G. Nilius B. J. Gen. Physiol. 2003; 121: 245-260Crossref PubMed Scopus (136) Google Scholar, 25Nilius B. Vennekens R. Prenen J. Hoenderop J.G. Droogmans G. Bindels R.J. J. Biol. Chem. 2001; 276: 1020-1025Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). As this negatively charged residue is most likely part of the selectivity filter, we tested whether mutations at this site influence the permeability of TRPV6 for large organic cations. Fig. 1B shows current-voltage relations with different extracellular monovalent cations for a mutant channel in which Asp541 was substituted by an alanine (D541A). In contrast to wild type TRPV6, the D541A mutant supported clear inward currents carried by TriMA+ and TetMA+. From the reversal potentials, we obtained PX/PNa values of 0.22 ± 0.02 and 0.08 ± 0.01 for TriMA+ and TetMA+, respectively. Likewise, introducing a glycine at this position (mutant D541G) also made the pore permeable to TriMA+ and TetMA+, with PX/PNa values of 0.27 ± 0.03 and 0.15 ± 0.02, respectively. Both D541A and D541G displayed negligible permeability to NMDG+ (PNMDG/PNa < 0.01). Introduction of an asparagine at this position (mutant D541N) did not significantly alter the permeability of the channel to the ammonium derivatives; TriMA+ and TetMA+ did not display measurable permeability, whereas the PX/PNa values for A+, MA+, and DMA+ were not significantly different from WT TRPV6 (Fig. 1C).Fig. 1D plots the permeability ratios of the different cations versus their estimated diameter for WT TRPV6 and the D541A, D541G, and D541N mutants. Data points were fitted using the excluded volume Equation 1 (26Dwyer T.M. Adams D.J. Hille B. J. Gen. Physiol. 1980; 75: 469-492Crossref PubMed Scopus (268) Google Scholar), PX/PNa=k(1-a/d)2/a(Eq. 1) where a is the cation diameter, and d the minimal pore diameter. The fit yielded a pore diameter of 5.4 Å for WT TRPV6. The same value was found for the D541N mutant, whereas significantly larger pore diameter estimates of 6.4 and 6.6 Å were obtained for the D541A and D541G mutants, respectively. Thus, replacement of Asp541 by an alanine or glycine, which have a shorter side chain, induces an apparent enlargement of the pore diameter by ∼1 Å. Replacement by an asparagine, whose side chain is of similar length as that of an aspartate, does not influence the pore diameter. These data demonstrate that Asp541 is an important determinant of the molecular sieving properties of TRPV6 and indicate that the amino acid side chain at this position contributes to the narrowest part of the selectivity filter.Accessibility of the Pore Region to Ag+—Next, we performed a SCAM analysis to identify amino acids accessible to extracellularly applied water-soluble cysteine reagents. We concentrated on residues Pro526 to Ala547 in the TM5-TM6 linker (Fig. 2), as this stretch of amino acids displays the highest degree of sequence homology with the bacterial K+ channel KcsA, in a region that forms the pore helix and selectivity filter. Cysteine substitutions were well tolerated in the region between Pro526 and Ile539; with the exception of L537C, all mutants in this region functionally expressed as Ca2+-selective channels and became permeable to monovalent cations upon omission of extracellular divalent cations. Mutants N545C and N547C also expressed as a functional cation channel. In contrast, mutants with cysteines introduced at positions 540–544 and 546 were non-functional.Fig. 2Alignment of the putative pore region of TRPV6 with the pore region of the K+ channel KcsA. Identical and conserved residues are marked with asterisks or dots, respectively. Regions that constitute the pore helix and selectivity filter in the KcsA crystal structure (18Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5688) Google Scholar) are also indicated. Accession numbers for mouse TRPV6 and KcsA (from Streptomyces lividans) are CAD62684 and S60172, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We first tested the functional cysteine mutants for their sensitivity to extracellularly applied Ag+. Chemically, Ag+ is a soft Lewis acid that reacts rapidly with the thiolate group of the cysteine side chain to form a strong covalent S–Ag bond (27Dance I.G. Polyhedron. 1986; 8: 1037-1104Crossref Scopus (741) Google Scholar). The diameter of Ag+ (a = 2.52 Å) is between that of Na+ (a = 1.90 Å) and K+ (a = 2.66 Å), two cations that readily permeate TRPV6 in the absence of extracellular divalent cations, suggesting that Ag+ can access the narrowest parts of the pore. We could discriminate four different types of Ag+ sensitivity. First, monovalent currents through wild type TRPV6 (Fig. 3A) and certain mutants (e.g. F536C; Fig. 3B) were virtually insensitive, showing less than 5% change in current amplitude after 200 s of treatment with 200 nm Ag+. Second, mutants such as S531C (Fig. 3C) reacted rapidly with Ag+ at a concentration of only 20 nm, resulting in a full block of monovalent currents within 10 s. Third, mutants such as L535C (Fig. 3D) clearly reacted with Ag+ but at a much slower rate. Finally, mutants P526C and M527C (Fig. 3E) displayed a clear biphasic response to Ag+.Fig. 3Effect of extracellular Ag+ on WT TRPV6 and cysteine mutants. A–F, time course of the inward current at -80 mV showing the effect of the indicated concentrations of free Ag+ on the whole-cell current through WT TRPV6 and the indicated cysteine mutants.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In order to obtain functional expression of the silent cysteine mutants, we coexpressed them together with WT TRPV6. This approach has previously been successfully used to obtain functional Shaker K+ channels with cysteines introduced at critical sites in the pore (28Lu Q. Miller C. Science. 1995; 268: 304-307Crossref PubMed Scopus (172) Google Scholar). No sizeable currents could be measured from cells coexpressing WT TRPV6 and either L537C, D541C, or Y546C, indicating that cysteines introduced at these positions have a dominant negative effect on channel activity. Alternatively, these mutants might somehow interfere with the targeting of functional channels to the plasma membrane. In contrast, robust currents could be measured from cells expressing WT TRPV6 together with the four other silent mutants (I540C, G542C, P543C, and A544C). In all four cases, these currents were sensitive to Ag+ (e.g. A544C; Fig. 3F), demonstrating the reaction between Ag+ and cysteine residues in the context of a functional heterotetrameric channel consisting of both WT and mutant subunits.Fig. 4A summarizes the inhibitory effects of Ag+ on whole-cell currents through WT TRPV6 and the different mutant channels. To further quantify the reactivity of the introduced cysteines, we calculated the second-order reaction rate constant for inhibition by Ag+ according to Equation 2, kAg=1/([Ag+]×τ)(Eq. 2) where τ is the time constant obtained by fitting an exponential function to the time course of current inhibition by Ag+ (Fig. 4B). Two kAg values are shown for P526C and M527C, corresponding to the fast and slow phase of Ag+ inhibition. For mutants that did not show significant inhibition after treatment with 200 nm Ag+ for 200 s, kAg was set to zero. Note that the fastest rates (>107m-1 s-1) may be an underestimate of the actual reaction speed, as ∼1 s was needed to fully replace the bathing solution surrounding the patch-clamped cell with Ag+-containing solution.Fig. 4Summary of the Ag+ reactivity of TRPV6 cysteine mutants. A, percentage inhibition of the inward Na+ current through WT TRPV6 and the indicated mutants after exposure to 200 nm free Ag+ for 200 s. Dashed bars indicate mutants that only yielded currents when coexpressed with WT TRPV6. Question marks indicate cysteine mutants that failed to express as functional ion channels. Inhibition by Ag+ was statistically significant (p < 0.05) in all cases, except for WT, L529C, T532C, and F536C. B, reaction rates for the modification of the different mutant channels by Ag+. Two reaction rates are indicated for mutants P526C and M527C, reflecting the double exponential time course of Ag+ block. C, helical wheel representation of residues Pro526 to Thr538, assuming a classical α-helix with 3.6 residues per turn. Filled circles correspond to residues at which substituted cysteine reacts rapidly with Ag+ (reaction rate >5 × 106m-1 s-1); half-open circles correspond to less reactive substituted cysteines (reaction rate <5 × 106m-1 s-1), and open circles correspond to non-reactive substituted cysteines.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the crystal structure of KcsA, residues 63–75 form an α-helical structure, which constitutes the pore helix (18Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5688) Google Scholar). Fig. 4C shows a helical wheel representation of the corresponding residues in TRPV6 (Pro526 to Thr538), assuming a classical α-helical structure with 3.6 amino acids per turn. Strikingly, all residues that rapidly react with Ag+ are on the same side of the helix, whereas the non-exposed residues are clustered on the opposite side. From these data we infer that residues Pro526 to Thr538 form an α-helical pore helix.Accessibility of the Pore Region to Cationic MTS Reagents— The positively charged MTSEA and MTSET attach, respectively, -SCH2CH2NH)3+ or -SCH2CH2N(CH3)3+ groups to free sulfhydryls (19Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (542) Google Scholar). MTSEA and MTSET have a more or less cylindrical shape and contain polar heads that are 3.6 and 5.8 Å in diameter, respectively. To investigate the accessibility of substituted cysteines residues in the pore region to these larger modifying agents, we applied MTSEA and MTSET to the bath solution and looked for modification of the monovalent current through WT TRPV6 and the different cysteine mutants.Currents through WT TRPV6 (not shown) and certain mutants (e.g. F536C; Fig. 5A) were virtually insensitive to MTSEA or MTSET, showing less than 5% change in current amplitude after 200 s of treatment with 1 mm of these reagents. Other mutants (e.g. F530C; Fig. 5B) reacted rapidly with both MTSEA and MTSET, resulting in a complete and irreversible block of the current. Fig. 5E" @default.
• W2023463047 created "2016-06-24" @default.
• W2023463047 creator A5021952303 @default.
• W2023463047 creator A5071455949 @default.
• W2023463047 creator A5087089940 @default.
• W2023463047 creator A5088168092 @default.
• W2023463047 date "2004-04-01" @default.
• W2023463047 modified "2023-10-10" @default.
• W2023463047 title "Outer Pore Architecture of a Ca2+-selective TRP Channel" @default.
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