Molecular Determinants of Permeation through the Cation Channel TRPV4
We have studied the molecular determinants of ion permeation through the TRPV4 channel (VRL-2, TRP12, VR-OAC, and OTRPC4). TRPV4 is characterized by both inward and outward rectification, voltage-dependent block by Ruthenium Red, a moderate selectivity for divalent versus monovalent cations, and an Eisenman IV permeability sequence. We identify two aspartate residues, Asp672 and Asp682, as important determinants of the Ca2+ sensitivity of the TRPV4 pore. Neutralization of either aspartate to alanine caused a moderate reduction of the relative permeability for divalent cations and of the degree of outward rectification. Neutralizing both aspartates simultaneously caused a much stronger reduction of Ca2+permeability and channel rectification and additionally altered the permeability order for monovalent cations toward Eisenman sequence II or I. Moreover, neutralizing Asp682 but not Asp672 strongly reduces the affinity of the channel for Ruthenium Red. Mutations to Met680, which is located at the center of a putative selectivity filter, strongly reduced whole cell current amplitude and impaired Ca2+ permeation. In contrast, neutralizing the only positively charged residue in the putative pore region, Lys675, had no obvious effects on the properties of the TRPV4 channel pore. Our findings delineate the pore region of TRPV4 and give a first insight into the possible architecture of its permeation pathway. We have studied the molecular determinants of ion permeation through the TRPV4 channel (VRL-2, TRP12, VR-OAC, and OTRPC4). TRPV4 is characterized by both inward and outward rectification, voltage-dependent block by Ruthenium Red, a moderate selectivity for divalent versus monovalent cations, and an Eisenman IV permeability sequence. We identify two aspartate residues, Asp672 and Asp682, as important determinants of the Ca2+ sensitivity of the TRPV4 pore. Neutralization of either aspartate to alanine caused a moderate reduction of the relative permeability for divalent cations and of the degree of outward rectification. Neutralizing both aspartates simultaneously caused a much stronger reduction of Ca2+permeability and channel rectification and additionally altered the permeability order for monovalent cations toward Eisenman sequence II or I. Moreover, neutralizing Asp682 but not Asp672 strongly reduces the affinity of the channel for Ruthenium Red. Mutations to Met680, which is located at the center of a putative selectivity filter, strongly reduced whole cell current amplitude and impaired Ca2+ permeation. In contrast, neutralizing the only positively charged residue in the putative pore region, Lys675, had no obvious effects on the properties of the TRPV4 channel pore. Our findings delineate the pore region of TRPV4 and give a first insight into the possible architecture of its permeation pathway. transient receptor potential human embryonic kidney 4α -phorbol 12,13-didecanoate Ruthenium Red bis(O-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid The TRPV subfamily of transient receptor potential (TRP)1 proteins homologous to the vanilloid receptor consists of at least five mammalian Ca2+-permeable cation channels, which are activated by a variety of signals including noxious chemical and thermal stimuli (TRPV1 (VR1) and TRPV2 (VRL-1)), increased cell volume (TRPV4 (VR-OAC, OTRPC4, and TRP12)), or decreased intracellular Ca2+ (TRPV5 (ECaC1) and TRPV6 (ECaC2)) (Ref. 1Harteneck C. Plant T.D. Schultz G. Trends Neurosci. 2000; 23: 159-166Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar; for a unifying nomenclature see Ref. 2Montell C. Birnbaumer L. Flockerzi V. Bindels R.J. Bruford E.A. Caterina M.J. Clapham D. Harteneck C. Heller S. Julius D. Kojima I. Mori Y. Penner R. Prawitt D. Scharenberg A.M. Schultz G. Shimizu S. Zhu M.X. Mol. Cell. 2002; 9: 229-231Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). TRPV channels typically contain three to six ankyrin repeats in the N terminus, six transmembrane segments with a putative pore region between transmembrane segments 5 and 6 but lack the so-called TRP motif (1Harteneck C. Plant T.D. Schultz G. Trends Neurosci. 2000; 23: 159-166Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 2Montell C. Birnbaumer L. Flockerzi V. Bindels R.J. Bruford E.A. Caterina M.J. Clapham D. Harteneck C. Heller S. Julius D. Kojima I. Mori Y. Penner R. Prawitt D. Scharenberg A.M. Schultz G. Shimizu S. Zhu M.X. Mol. Cell. 2002; 9: 229-231Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). Recent findings indicate that TRPV4, which was originally identified as an osmotically activated channel (3Wissenbach U. Bodding M. Freichel M. Flockerzi V. FEBS Lett. 2000; 485: 127-134Crossref PubMed Scopus (257) Google Scholar, 4Strotmann R. Harteneck C. Nunnenmacher K. Schultz G. Plant T.D. Nat. Cell Biol. 2000; 2: 695-702Crossref PubMed Scopus (805) Google Scholar, 5Liedtke W. Choe Y. Marti Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1091) Google Scholar), is not only activated by mechanical stimuli but also by ligands such as phorbol derivatives (6Watanabe H. Davis J.B. Smart D. Jerman J.C. Smith G.D. Hayes P. Vriens J. Cairns W. Wissenbach U. Prenen J. Flockerzi V. Droogmans G. Benham C.D. Nilius B. J. Biol. Chem. 2002; 277: 13569-13577Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar). Therefore, the functional importance of this channel may be connected to its role as a promiscuous Ca2+ influx channel integrating multiple physical and chemical stimuli. Based on their pore properties, the TRPV subfamily can be subdivided into two groups. TRPV5 and TRPV6, on the one hand, are highly Ca2+ selective channels displayingPCa/PNa values of more than 100 and a monovalent cation permeability sequence corresponding to a strong field binding site (Eisenman X or XI) (7Voets T. Prenen J. Fleig A. Vennekens R. Watanabe H. Hoenderop J.G.J. Bindels R.J.M. Droogmans G. Penner R. Nilius B. J. Biol. Chem. 2001; 276: 47767-47770Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 8Yue L. Peng J.B. Hediger M.A. Clapham D.E. Nature. 2001; 410: 705-709Crossref PubMed Scopus (320) Google Scholar, 9Vennekens 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 (274) Google Scholar, 10Nilius B. Vennekens R. Prenen J. Hoenderop J.G. Bindels R.J. Droogmans G. J. Physiol. (Lond.). 2000; 1: 2239-2248Google Scholar). It has been demonstrated that their high Ca2+ selectivity crucially depends on a single negatively charged aspartate residue in the pore region (11Nilius B. Vennekens R. Prenen J. Hoenderop J.G.J. Droogmans G. Bindels R.J.M. J. Biol. Chem. 2001; 276: 1020-1025Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). On the other hand, TRPV1, TRPV2, and TRPV4 are only weakly Ca2+-selective withPCa/PNa values below 10 (4–6, 12–14), and their monovalent cation permeability sequences are more indicative of a weak field strength binding site (6Watanabe H. Davis J.B. Smart D. Jerman J.C. Smith G.D. Hayes P. Vriens J. Cairns W. Wissenbach U. Prenen J. Flockerzi V. Droogmans G. Benham C.D. Nilius B. J. Biol. Chem. 2002; 277: 13569-13577Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, 12Caterina M.J. Rosen T.A. Tominaga M. Brake A.J. Julius D. Nature. 1999; 398: 436-441Crossref PubMed Scopus (1264) Google Scholar, 13Caterina M.J. Schumacher M.A. Tominaga M. Rosen T.A. Levine J.D. Julius D. Nature. 1997; 389: 816-824Crossref PubMed Scopus (7144) Google Scholar, 14Nilius B. Prenen J. Wissenbach U. Bo¨dding M. Droogmans G. Pflu¨gers Arch. Eur. J. Physiol. 2001; 443: 227-233Crossref PubMed Scopus (119) Google Scholar). The precise molecular basis for these divergent pore properties is not yet fully understood. In the present study, we were led by a sequence comparison of the pore region of the TRPVs to search for possible molecular determinants for the pore features of TRPV4 and identify several residues that contribute to the permeability profile and blocker sensitivity of the channel. We used the recombinant bicistronic expression plasmid pdiTRP12, which carries the entire protein-coding region for murine TRPV4 (mouse mTRP12; accession numberCAC20703) and for green fluorescent protein coupled by an IRES sequence. Human embryonic kidney cells, HEK293, were grown in Dulbecco’s modified Eagle’s medium containing 10% (v/v) human serum, 2 mml-glutamine, 2 units/ml penicillin, and 2 mg/ml streptomycin at 37 °C in a humidity controlled incubator with 10% CO2. HEK293 cells were transiently transfected with the above-described vector using methods described previously (9Vennekens 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 (274) Google Scholar). Positively transfected cells were visually identified based on their green fluorescence. Nontransfected cells from the same batch were used as controls. Mutations to the TRPV4 pore were made using the QuikChangeTM site-directed mutagenesis kit (Stratagene). The nucleotide sequences of the mutants have been verified by sequencing of the corresponding cDNAs. The standard extracellular solution contained 150 mm NaCl, 1 mm MgCl2, 5 mm CaCl2, 10 mm glucose, 10 mm HEPES, buffered at pH 7.4 with NaOH. The osmolality of this solution, as measured with a vapor pressure osmometer (Wescor 5500, Schlag, Gladbach, Germany), was 320 ± 5 mosmol. When indicated in the figure legends, the Ca2+ concentration of this solution was varied between 0 and 30 mm. To study the relative permeability of mono- and divalent cations, we used extracellular solutions containing 1 mm MgCl2, 10 mm glucose, 10 mm HEPES, and either 150 mmXCl (where X = sodium, lithium, cesium, potassium, or rubidium) or 30 mmXCl2 (X = calcium or magnesium) and 120 N-methyl-d-glucamine chloride. These solutions were titrated to pH 7.4 with the appropriate base. Two different intracellular solutions were used yielding virtually identical results after correcting for the liquid junction potential (see below). The cesium-based solution contained 20 mmCsCl, 100 mm cesium aspartate, 1 mmMgCl2, 4 mm Na2ATP, 0.022 mm CaCl2, 10 mm BAPTA, 10 mm HEPES, pH adjusted to 7.2 with CsOH. The solution contained 150 mm NaCl, 1 mmMgCl2, 4 mm Na2ATP, mm CaCl2, 5 mm 10 mm HEPES, pH adjusted to 7.2 with NaOH. intracellular Ca2+ was to be for both The TRPV4 12,13-didecanoate (6Watanabe H. Davis J.B. Smart D. Jerman J.C. Smith G.D. Hayes P. Vriens J. Cairns W. Wissenbach U. Prenen J. Flockerzi V. Droogmans G. Benham C.D. Nilius B. J. Biol. Chem. 2002; 277: 13569-13577Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar), a phorbol was to the extracellular solution at a concentration of 1 from 30 mm solutions in cell were with an filter, using had a between 2 and 4 with intracellular was used as a and were and of the was to we have a of a to by a to was 5 The were at The of the whole cell current and current were by the current in a the of the were at The relative permeability of monovalent cations was from the in potential after of extracellular by the to the 1 is the measured in of the divalent cations Ca2+ and relative to was from the potential measured with 30 mm of the cation in the extracellular solution, to the the permeability of the divalent its extracellular from and are the and intracellular for and and the potential (9Vennekens 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 (274) Google Scholar, 10Nilius B. Vennekens R. Prenen J. Hoenderop J.G. Bindels R.J. Droogmans G. J. Physiol. (Lond.). 2000; 1: 2239-2248Google Scholar, B. Vennekens R. Prenen J. Hoenderop J.G.J. Droogmans G. Bindels R.J.M. J. Biol. Chem. 2001; 276: 1020-1025Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). of the relative were for liquid junction PubMed Scopus Google the liquid junction was to P.H. J. Neurosci. PubMed Scopus Google Scholar). that the in 5 are not for liquid junction of pore on Ca2+permeability of of through fully activated and TRPV4 in standard extracellular solution and in an extracellular solution containing 30 mm Ca2+ as the In the in potential is The values are in I. and of the potential for the and permeability sequence is altered in the The monovalent through and TRPV4 in extracellular solutions containing 150 mm of the indicated monovalent cation as are the different permeability sequence for the rectification of and for TRPV4 in standard extracellular solution containing the indicated Ca2+ the of inward and outward rectification in the of same as in for the current for TRPV4 and three pore mutants measured with 5 mm extracellular The were to the current at of rectification properties for and are the of the measured at and The are as the ± was by of In of were using an of the putative pore region of murine TRPV4 with the corresponding of the other of the TRPV subfamily and of the channel In this region, TRPV4 has the with and TRPV2, with which a of highly is also with TRPV5 and TRPV6 to a with the pore region of When with the highly Ca2+-selective two are the contain a positively charged which is not present in contain a in at the corresponding to in TRPV5 in which is for the high Ca2+ selectivity of the channel. Moreover, TRPV4 two negatively charged residues the of which is and Asp682, which is not present in and to in TRPV1, a residue in Ruthenium Red block and To the of these TRPV4 pore residues to the pore properties of the we for TRPV4 channels in which were into the pore. residues were and and the pore residue was by an alanine to the a was either or in with a of the positively charged and in mutants as functional ion channels, as indicated by the of a cation current of transfected HEK293 cells with the TRPV4 When with TRPV4, the current were for the and to led to a reduction in current These in current from in or sensitivity toward by not be of by extracellular depends on a negatively charged residue the pore region Tominaga M. Julius D. U. S. A. 2000; PubMed Scopus Google Scholar), but more is to a possible of pore residues in of In this we on the of these residues to pore rectification, and voltage-dependent It has been previously that TRPV4 is selective for Ca2+ and a monovalent cation permeability sequence corresponding to Eisenman We these properties are by to the putative pore region by the relative cation for of the current with we between extracellular solutions containing a single cation and relative from the in the potential after correcting for liquid junction (see and TRPV4, from a extracellular solution to solutions containing 30 mm Ca2+ or as the cation caused a of the inward current at and a of the potential to were ± = and ± for Ca2+ and extracellular by other monovalent cations caused only in the potential that TRPV4 between monovalent The monovalent cation permeability sequence was which to Eisenman sequence IV for a weak field strength possible for the and the weak field strength of the TRPV4, TRPV1, and TRPV2 be the of a positively charged in in the of the putative pore region, which is not present in the strong field strength site TRPV5 and TRPV6 neutralizing this into alanine had no on the divalent selectivity of the channel the pore between monovalent cations and the Eisenman IV permeation the field strength binding site is that not the field the TRPV4 channel pore. The two negatively charged residues in the pore region, Asp672 and Asp682, as the TRPV4 to the role by aspartate and residues in the Ca2+ selective of TRPV6, and Ca2+ with such a we that the and mutants both a reduced as can be from the to a extracellular solution and The relative Ca2+ permeability was in both to ± = and ± = for and inward current with 30 mm Ca2+ as the were for both mutants than for TRPV4 the current in solutions that Ca2+ is these pore aspartates are TRPV4, the and between monovalent cations and the Eisenman IV permeation profile and not we that both channels a relative permeability for values of ± = and ± = with ± = for TRPV4 of and TRPV4 values in indicate the of a selectivity sequence was and the of ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± inward and strong of the indicative of a ± ± ± ± II ± ± ± ± ± ± inward and strong of the indicative of a inward and strong of the indicative of a ± ± ± ± inward and strong of the indicative of a inward and strong of the indicative of a ± ± ± ± relative were as described and The are as the ± from at least three The values in indicate the of a selectivity sequence was and the of inward and strong of the indicative of a in a The relative were as described and The are as the ± from at least three of both aspartates more in permeation The relative Ca2+ permeability was more reduced than in the single mutants = ± = and was no a permeability for Moreover, the permeability sequence for monovalent cations was different from that of with both and more than in of cells The permeability sequence is with a reduction of the field strength of the site to Eisenman sequence II or We also important in the rectification properties between TRPV4 and the as be from in Ca2+ the sequence comparison in two of indicated that is at a the putative pore region of at the corresponding in TRPV5 we an aspartate which is for the high Ca2+ selectivity of this channel. at the corresponding in channels is a of the so-called channel contribute to the of the selectivity in which was by alanine or aspartate to but strongly reduced current with which may have on the of relative of the and in divalent permeability of these mutants were In the from a extracellular solution a solution containing 30 mm Ca2+ as the cation caused a of the potential corresponding to of ± = the same we a of the potential for the and mutants that these pore the Ca2+ selectivity of the TRPV4 pore values with TRPV4, no in the monovalent permeability sequence (Eisenman were not but three mutants had a relative permeability for The of TRPV4 in extracellular solutions containing of mono- and divalent cations both inward and outward rectification. in this strongly depends on the extracellular Ca2+ In the of divalent cations, the is with identical current at and of Ca2+ an of the current that is more at of a voltage-dependent to 30 mm not a block of the inward Ca2+ the as by the of the To the of extracellular Ca2+ we the inward current at to the outward current at that the Ca2+ block at highly TRPV4, decreased from in solution to in the of 30 mm identical results were for the that this residue is not an important of TRPV4 pore properties and not In contrast, of Asp672 and Asp682 strongly reduced the sensitivity of the channel to extracellular In the the was with 1 in the extracellular solution and outward rectification reduced for to 30 mm The rectification properties of the single mutants and were between and the which can also be from the measured with 5 mm extracellular Ca2+ These results indicate that both aspartate residues contribute to the Ca2+ sensitivity of the TRPV4 pore. Ruthenium Red is a that has been to of the TRPV with to in and extracellular is a voltage-dependent blocker of a concentration of 1 extracellular inward outward were measured at more than and The of channels, measured from increased from at to at with block at ± = The of block was with a for block of ± at = We also in which a high concentration of was in the this we not of the TRPV4 a of 1 to the extracellular medium in the voltage-dependent These that TRPV4 from the extracellular by binding in a voltage-dependent to a site in the channel the transmembrane identical results were for the and mutants and not In contrast, the and mutants were much to block by extracellular and and not and the of the block was to more both the of channels increased from at to at with block at ± = and ± = for and The of block was also much for these with of ± = and ± = the of Asp682 to be for the high sensitivity of TRPV4 for to in the pore of C. C. R. J.M. A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). In the of the TRP of cation channels has of Ca2+ influx in cell is not the that are after expression of of the TRP are by the TRP proteins or as of The is by the findings that TRP proteins can C. G. L. Clapham D.E. 2001; Full Text Full Text PDF PubMed Scopus Google in intracellular P. Y. L. Y. S. R. S. Nat. Cell Biol. 2002; PubMed Scopus Google Scholar). The of channel permeation properties and of these properties by to pore residues the of the channel of these this has only been for and TRPV5 (11Nilius B. Vennekens R. Prenen J. Hoenderop J.G.J. Droogmans G. Bindels R.J.M. J. Biol. Chem. 2001; 276: 1020-1025Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, C. C. R. J.M. A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. P.H. U. S. A. 2000; PubMed Scopus Google Scholar). The present study of the TRPV to this the and We have demonstrated that two negatively charged aspartate residues and the acid are determinants of the pore properties of In the of both monovalent and divalent cations in the extracellular solution, the of TRPV4 both inward and outward rectification, a with TRPV1, TRPV2, and several of the and D.E. C. Nat. Neurosci. 2001; 2: PubMed Scopus Google Scholar). Our present results at least for TRPV4, this depends on extracellular Ca2+ which both block the channel in a voltage-dependent and the channel. We that two negatively charged residues, Asp672 and Asp682, contribute to the as by the reduced rectification and relative Ca2+ permeability in both the and The an stronger reduction in rectification and Ca2+ that the of neutralizing these aspartates on the Ca2+ sensitivity of TRPV4 is Moreover, this has a selectivity for monovalent cations and a indicative of a weak field binding site (Eisenman or These results to indicate that these aspartates are fully and contribute to a single negatively charged binding site for cations the channel pore. both aspartates contribute to the voltage-dependent block of TRPV4 by the the is with the same affinity and as by TRPV4, block of the or mutants is strongly The potential for block of these mutants by 1 is to the by and the of block at is these results indicate that both aspartates contribute to the of the TRPV4 pore but in their to extracellular Asp682 is to the of the can with both Ca2+ and In contrast, Asp672 is to be located more toward the site of the can with the cation Ca2+ but not with the cation Our results also indicate that is for the of the TRPV4 pore. this residue with an alanine results in a decreased Ca2+ permeability and current a at this a reduction of the current and a of Ca2+ permeation. possible is that this the affinity of the site to such an that can no the channel. We had that of the positively charged residue be in the field strength of the putative site the pore. from the that this residue is not present in the highly Ca2+-selective channels TRPV5 and permeation properties for monovalent cations are not and the relative Ca2+ permeability in the the two other mutants that were to the high Ca2+ selectivity and strong field binding site of TRPV4 and not give the and channels with strongly reduced Ca2+ It that the in pore properties between and be reduced to single acid the pore in the pore region of TRPV4 with the pore region of the channel has been at Y. A. R. Nature. 2001; PubMed Scopus Google J. A. J.M. R. PubMed Scopus Google Scholar). the that both channels a pore The results from mutagenesis study are at least with this as we that Asp682 is located at the extracellular of the TRPV4 pore can with Asp672 to be located more toward the in the to Asp682 in is located at the of the selectivity filter, to is more toward the intracellular of the channel. we that is for channel to an alanine or aspartate strongly the permeability for monovalent and divalent In the the corresponding is at the center of the selectivity filter, its with Y. A. R. Nature. 2001; PubMed Scopus Google Scholar, J. A. J.M. R. PubMed Scopus Google Scholar). we that the positively charged not the pore properties of a at the corresponding the between and TRPV4 is to be of the so-called pore J. A. J.M. R. PubMed Scopus Google Scholar), the residues of which not contribute to the of the channel pore. In we have that TRPV4 is a protein that to in its putative pore region with in ion permeability and Two negatively charged aspartates and a residue were identified as determinants of the pore properties of the channel. Moreover, results are with the that TRPV channels have the same pore architecture as including a of the of the may be to this The present may also a for at the of the or the of more selective TRPV4 We Flockerzi the of the and