Voltage Dependence of the Ca2+-activated Cation Channel TRPM4

Abstract

TRPM4 is a Ca2+-activated but Ca2+-impermeable cation channel. An increase of [Ca2+]i induces activation and subsequent reduction of currents through TRPM4 channels. This inactivation is strikingly decreased in cell-free patches. In whole cell and cell-free configuration, currents through TRPM4 deactivate rapidly at negative potentials. At positive potentials, currents are much larger and activate slowly. This voltage-dependent behavior induces a striking outward rectification of the steady state currents. The instantaneous current-voltage relationship, derived from the amplitude of tail currents following a prepulse to positive potentials, is linear. Currents show a Boltzmann type of activation; the fraction of open channels increases at positive potentials and is low at negative potentials. Voltage dependence is not due to block by divalent cations or to voltage-dependent binding of intracellular Ca2+ to an activator site, indicating that TRPM4 is a transient receptor potential channel with an intrinsic voltage-sensing mechanism. Voltage dependence of TRPM4 may be functionally important, especially in excitable tissues generating plateau-like or bursting action potentials. TRPM4 is a Ca2+-activated but Ca2+-impermeable cation channel. An increase of [Ca2+]i induces activation and subsequent reduction of currents through TRPM4 channels. This inactivation is strikingly decreased in cell-free patches. In whole cell and cell-free configuration, currents through TRPM4 deactivate rapidly at negative potentials. At positive potentials, currents are much larger and activate slowly. This voltage-dependent behavior induces a striking outward rectification of the steady state currents. The instantaneous current-voltage relationship, derived from the amplitude of tail currents following a prepulse to positive potentials, is linear. Currents show a Boltzmann type of activation; the fraction of open channels increases at positive potentials and is low at negative potentials. Voltage dependence is not due to block by divalent cations or to voltage-dependent binding of intracellular Ca2+ to an activator site, indicating that TRPM4 is a transient receptor potential channel with an intrinsic voltage-sensing mechanism. Voltage dependence of TRPM4 may be functionally important, especially in excitable tissues generating plateau-like or bursting action potentials. Voltage dependence of the Ca2+-activated cation channel TRPM4.Journal of Biological ChemistryVol. 278Issue 43PreviewVol. 278 (2003) 30813-30820 Full-Text PDF Open Access Cation channels of the transient receptor potential (TRP) 1The abbreviations used are: TRP, transient receptor potential; TM, transmembrane-spanning helix; DVF, divalent cation-free solution(s); HEDTA, N-hydroxylethyl-ethylenediamine-triacetic acid.1The abbreviations used are: TRP, transient receptor potential; TM, transmembrane-spanning helix; DVF, divalent cation-free solution(s); HEDTA, N-hydroxylethyl-ethylenediamine-triacetic acid. superfamily have received much attention during the last years because of their pivotal role in various cell functions linked to the modulation of intracellular Ca2+ signals, mostly in nonexcitable cells (1Montell 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 (559) Google Scholar, 2Montell C. Birnbaumer L. Flockerzi V. Cell. 2002; 108: 595-598Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar, 3Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (956) Google Scholar). More than 20 mammalian TRP members are known, which are classified into the TRPC (C for “canonical”), TRPV (V for “vanilloid”), and TRPM (M for “melastatin”) subfamilies (1Montell 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 (559) Google Scholar, 2Montell C. Birnbaumer L. Flockerzi V. Cell. 2002; 108: 595-598Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar). However, the functional properties of most members of this novel and fast growing channel family are not yet analyzed in detail. The predicted transmembrane topology of TRPs is similar to that of voltage-gated and cyclic nucleotide gated channels; they consist of six transmembrane-spanning helices (TM1 to -6), cytoplasmic N and C termini, and a pore region between TM5 and TM6 (2Montell C. Birnbaumer L. Flockerzi V. Cell. 2002; 108: 595-598Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar, 3Clapham D.E. Runnels L.W. Strubing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (956) Google Scholar). Since the fourth transmembrane helix is not positively charged, TRP channels were considered as voltage-independent. The voltage-sensing features of some members of the TRPV subfamily could be attributed to voltage-dependent block of the channel pore by intra- or extracellular divalent cations (4Voets T. Janssens A. Prenen J. Droogmans D. Nilius G. J. Gen. Physiol. 2003; 121: 245-260Crossref PubMed Scopus (137) Google Scholar, 5Voets 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). Members of the TRPM subfamily are much less studied at the functional level than those of the TRPV and TRPC family. They are characterized by relatively long N and C termini, and some of them have entire enzyme domains linked to their C termini: an ADP-ribose pyrophosphatase in TRPM2 (6Perraud A.L. Fleig A. Dunn C.A. Bagley L.A. Launay P. Schmitz C. Stokes A.J. Zhu Q. Bessman M.J. Penner R. Kinet J.P. Scharenberg A.M. Nature. 2001; 411: 595-599Crossref PubMed Scopus (746) Google Scholar) and an atypical α-kinase domain in TRPM6 and TRPM7 (7Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (622) Google Scholar, 8Nadler M.J. Hermosura M.C. Inabe K. Perraud A.L. Zhu Q. Stokes A.J. Kurosaki T. Kinet J.P. Penner R. Scharenberg A.M. Fleig A. Nature. 2001; 411: 590-595Crossref PubMed Scopus (798) Google Scholar, 9Schlingmann K.P. Weber S. Peters M. Niemann Nejsum L. Vitzthum H. Klingel K. Kratz M. Haddad E. Ristoff E. Dinour D. Syrrou M. Nielsen S. Sassen M. Waldegger S. Seyberth H.W. Konrad M. Nat. Genet. 2002; 31: 166-170Crossref PubMed Scopus (647) Google Scholar, 10Yamaguchi H. Matsushita M. Nairn A.C. Kuriyan J. Mol. Cell. 2001; 7: 1047-1057Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 11Walder R.Y. Landau D. Meyer P. Shalev H. Tsolia M. Borochowitz Z. Boettger M.B. Beck G.E. Englehardt R.K. Carmi R. Sheffield V.C. Nat. Genet. 2002; 31: 171-174Crossref PubMed Scopus (470) Google Scholar). Surprisingly, TRPM4b, which is a Ca2+-impermeable monovalent cation channel of 25-picosiemens unitary conductance belonging to the TRPM subfamily, is in contrast to other TRP channels not inactivated but activated by intracellular Ca2+, [Ca2+]i (12Launay 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 (539) Google Scholar). A short form of TRPM4, TRPM4a, is characterized in less detail and displays completely different properties with regard to Ca2+ permeability and activation (13Xu X.Z. Moebius F. Gill D.L. Montell C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10692-10697Crossref PubMed Scopus (184) Google Scholar). In this study, we report cloning of the human and mouse TRPM4 cDNAs. Transcripts of these genes are expressed in heart, kidney, and endothelial cells, indicating that this channel plays a role in the cardiovascular system. We demonstrate that TRPM4 is a Ca2+- and voltage-dependent channel. It could therefore modulate the electrical activity of cells that generate action potentials. This is, to our knowledge, the first description of voltage-dependent properties of a TRP channel, suggesting a special role for this channel in excitable cells. Cloning of Human and Mouse TRPM4 cDNAs—The human expressed sequence tag 885075 (GenBank™), homologous to the human TRPM1 cDNA, was identified and sequenced on both strands; it contained a ∼1500-bp DNA fragment, which represented part of the TRPM4 cDNA. Northern and dot blot analysis using this DNA fragment as a probe revealed TRPM4 transcript expression in a number of tissues including human prostate. TRPM4 full-length cDNA clones were isolated from four independent oligo(dT)-primed and specifically primed cDNA libraries constructed with poly(A+) RNA isolated from tissue samples taken from patients with benign prostatic hyperplasia. Several cDNA clones (Fig. 1A) were identified and sequenced on both strands. The cDNAs obtained encoded two variants of the TRPM4 protein, a long variant of 1214 amino acids (calculated M r ∼134,000; GenBank™ accession number AX443227), identical to the TRPM4b sequence described by Launay et al. (12Launay 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 (539) Google Scholar) and the shorter variant TRPM4c but not the TRPM4a variant (13Xu X.Z. Moebius F. Gill D.L. Montell C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10692-10697Crossref PubMed Scopus (184) Google Scholar). The TRPM4c cDNA (accession number: AX443225) encodes 678 amino acid residues starting at Met537 of the TRPM4b variant. The TRPM4b cDNA was subcloned into the dicistronic pdi vector (14Wissenbach U. Bödding M. Freichel M. Flockerzi V. FEBS Lett. 2000; 485: 127-134Crossref PubMed Scopus (256) Google Scholar). To identify the mouse TRPM4, we searched the mouse genome ENSEMBL data base (available on the World Wide Web at www.ensembl.org) with the human sequence using the tblastN program. Two novel Ensembl genes of 5.27 and 9.06 kb (ENSMUSG00000038260 and ENSMUSG00000003866) were predicted. Analysis of these predicted gene loci and the surrounding genomic sequence by comparison of these genomic sequences with the human cDNA lead to the identification of exons corresponding to the exons in the human TRPM4 gene including the initiation and termination signals of translation. Primers M4_41 (5′-CTA GTC TAG AGG TGA GGA TAA GGC CAC CAG-3′) and M4_45 (5′-TGC TCT AGA GGC TGG AGG AAA TAC ACG G-3′) were derived from these exon sequences of the mouse TRPM4 gene (exon 1 and exon 25, respectively), which correspond to sequences in the 5′- and 3′-untranslated regions of human TRPM4, and cDNA fragments were amplified from murine heart. The amplified DNAs were subcloned, and the DNAs of several clones were sequenced on both strands. The 1213-amino acid sequence of the derived mTRPM4 protein corresponds to the human TRPM4b sequence. In addition, two shorter variants represented by several independent clones including pM4–27 and pM4–28 (Fig. 1B) were identified, which might represent splice variants. The amino acid sequence encoded by clones pM4-27 and pM4-28 start at Met187. The subsequent sequence of pM4-27 is identical with the one of mTRPM4 (clone pM4-26) and covers 1027 amino acid residues. The clone pM4-28 contains an insertion, which results in a shift of the reading frame and a premature stop codon. Accordingly, transcripts made from pM4-28 encode a further shortened protein variant, which is predicted to have 592 amino acids (Fig. 1C). The mouse TRPM4 gene is localized on chromosome 7.B2 in a region that is syntenic to human chromosome 19.q13.33, where the human TRPM4 gene is localized. Northern Analysis—A human multiple-tissue RNA blot (Clontech) was hybridized in the presence of 50% at The probe was the fragment of the human TRPM4 cDNA. were to for 20 the analysis of expression of TRPM4 in mouse RNA was isolated from mouse endothelial cells, heart, kidney, and cells as described (14Wissenbach U. Bödding M. Freichel M. Flockerzi V. FEBS Lett. 2000; 485: 127-134Crossref PubMed Scopus (256) Google Scholar). Mouse endothelial cells were isolated from as described Vennekens R. Freichel M. U. Prenen J. Flockerzi V. Droogmans G. Nilius B. J. Physiol. PubMed Scopus Google Scholar). Two fragments of and in corresponding to and of the mTRPM4 cDNA, were used as were to for to cells, were in human and at in a with of Human TRPM4 used the expression which the entire region for the human TRPM4b (accession and and for the protein by an sequence. cells were with the vector using described and cells were identified by their in the B. Vennekens R. Prenen J. Hoenderop Droogmans G. Bindels R.J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). extracellular for whole cell and the for contained and at with The for or the for whole contained 1 with Ca2+ at the of the was between and by of to by the (available on the at In some we used or of Ca2+ between and was to an were with Ca2+ at or with which identical results as in were were at cell currents were with an using patches. a between and An was used as a and were and of the was to channel data were obtained in the and The for channel was and was 1 for channel were from potentials between and of from to or by a to The between the was were with a prepulse to by from to of data cells were with the and in the and at of and through a at The at was at using a from the cell-free was was from the by [Ca2+]i 1 where is the binding is the at Ca2+, and 1 is that at were for the and the data were analyzed using the data are as of cells. was using The TRPM4 full-length cDNAs TRPM4b and murine were using RNA isolated from human tissue and mouse heart, The derived sequences of the human and mouse (Fig. sequence TRPM4 transcripts are expressed in most human and mouse tissues (Fig. including heart, and In human TRPM4 transcripts of and kb could be which represent the TRPM4 variants and isolated from the various cDNA libraries constructed from human transcripts could be in and The probe transcripts of were with human cDNA was expressed in cells in which we could not expressed TRPM4 transcripts by Northern blot and V. It in a report (12Launay 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 (539) Google Scholar) that TRPM4 is activated by an of the intracellular Ca2+ and the activated In our activation of TRPM4b by [Ca2+]i transient and by a which made it to A similar was not in cells or in cells using a indicating that activation on intracellular Ca2+ 20 an of this transient activated by the cell with 1 Ca2+ and the in [Ca2+]i (Fig. A and at the of the activated a (Fig. of TRPM4 currents is in the cell with Ca2+ activated a transient (Fig. inactivation of the to rapidly and the for several (Fig. This and is not due to a in [Ca2+]i because the the both at activation in whole cell and in both outward rectification (Fig. and The increase of the at negative potentials is the are from to but not they are from to and as be a of the at the potential by to negative potentials. of extracellular by completely currents (Fig. C and extracellular divalent cations cation-free not the (Fig. properties of currents through TRPM4 were further analyzed in cells with whole cell currents and currents in an from a cell with relatively of the in less than of the cells. currents inactivated completely the first from cells completely inactivated Currents activated by at a and in whole cell show the as in cell-free at and a at negative and activation at positive (Fig. the different in that at this the in whole cell was completely The was to in It inactivated and for several and features were identical to those of the whole cell (Fig. and activity was for than in to as by the channel in during to from a potential of channel were in cells or in cells in not The channel amplitude was at (Fig. corresponding to a channel conductance of a that is identical with (12Launay 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 (539) Google Scholar). This amplitude not by [Ca2+]i between and 1 not that the currents at from the corresponding channel the voltage-dependent as the The of at the of the is with a fast activation and of the channel (Fig. In C and the of currents from in divalent in the and in 1 and Currents were in the presence of Ca2+ for from to and and outward currents in much the currents in whole cell and and not the of the currents. Surprisingly, [Ca2+]i to activated channels in and in indicating that currents through TRPM4 not at as low as 1 as by Launay et al. (12Launay 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 (539) Google Scholar). We have activation of TRPM4 in the from to at 1 and these features were identical in (Fig. and properties of TRPM4 currents in whole cell and cell-free currents were activated in whole cell in with of the inactivation in this we were to the of the using at the a and that during the inactivation the amplitude at the of the in The the from the whole cell into the that the currents increase inactivation during the whole currents during from to potential 20 at the in A by The of the is and that in the of a fast at negative potentials and a activation at positive potentials. data are from the the different which the inactivation in whole cell but the relatively in the cell-free of TRPM4 in patches. channel from during to from a potential of The amplitude of the channel as from the corresponding amplitude was channel currents and from 20 for from to [Ca2+]i was currents from for to and the fast at negative and the activation at positive potentials. Ca2+ at the of the is was of divalent cations potential is as in C for a 1 and the between the in C and as as with the in the whole cell and the (Fig. dependence of the currents in the currents from the at different Ca2+ are at the of the that the currents increase by [Ca2+]i to and at to and as in from a with a 1 and of were to a at of whole cell and from to activation were whole and data that the voltage-dependent is identical for different The features in that the of TRPM4b is a voltage-dependent that be further in the following using (Fig. [Ca2+]i because the is in this Voltage from to negative potentials rapidly much larger outward currents were activated during to positive potentials (Fig. larger were this was from a potential of (Fig. which is with a larger fraction of channels at this positive The instantaneous obtained by the at the of as a of the are but at the potential were much than at (Fig. To the dependence of activation on the we used a similar as in and the amplitude during a to following the This induces which rapidly deactivate (Fig. from the currents at the of the outward rectification (Fig. The amplitude of the at the of the to a dependence on (Fig. The fraction of open channels at the of in was by these to the at positive potentials. It is that channel on potential and be described by a Boltzmann where open the fraction of open channels at the potential is the potential of is the and is the fraction of open channels at negative potentials (Fig. the data from cells at [Ca2+]i using of 1 and we obtained a steady state fraction of channels at negative potentials of a potential for activation of and a of To be that activation was at the of the to 1 and we the of activation using a of of by a to (Fig. The of the at could be by a with a of indicating that a steady state activation is at the of a in [Ca2+]i a on the amplitude of the activated currents (Fig. A and but not shift their (Fig. data from different for [Ca2+]i of and The data for were by a Boltzmann with the for for and for the potential of that the voltage-dependent of TRPM4b activation are not due to voltage-dependent binding of the intracellular Ca2+ divalent cations on the steady state it was not by divalent cations or from to for data not The TRPM4b channel is in as much as it is activated than inactivated by intracellular Ca2+ and is for Ca2+ (12Launay 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 (539) Google Scholar). We show that TRPM4b a striking which not described for TRP channel, indicating that TRPM4b is both a Ca2+-activated and voltage-dependent cation channel. The potential the channel but not activate the on an increase in TRPM4b in this from other Ca2+-activated as where lead to channel activity in the of intracellular In our activation of TRPM4b Ca2+ than those (12Launay 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 (539) Google Scholar). is for this In that the intracellular contained or but intracellular in the by or not the Ca2+ of Surprisingly, we in cells at at and inactivation the first into the cells. The of this is The of the is at suggesting a mechanism. This to be or in cell-free indicating that a might be This is at with data (6Perraud A.L. Fleig A. Dunn C.A. Bagley L.A. Launay P. Schmitz C. Stokes A.J. Zhu Q. Bessman M.J. Penner R. Kinet J.P. Scharenberg A.M. Nature. 2001; 411: 595-599Crossref PubMed Scopus (746) Google Scholar). The for these is not However, at Ca2+ in the 1 is and may not be in less than (6Perraud A.L. Fleig A. Dunn C.A. Bagley L.A. Launay P. Schmitz C. Stokes A.J. Zhu Q. Bessman M.J. Penner R. Kinet J.P. Scharenberg A.M. Nature. 2001; 411: 595-599Crossref PubMed Scopus (746) Google Scholar). It is to in with the modulation of the of binding of might be Julius D. Science. 2003; PubMed Scopus Google Scholar). could channel inactivation by the of Ca2+ of TRPM4b and the binding in a between residues and in TRPM4 that a with the in state activation potential of or indicating that a is The striking TRPM4b as a and voltage-dependent channel. features that TRPM4b could a functional in the modulation of action potentials, the transient currents during might the initiation of a subsequent This might be for and modulation of electrical activity or for modulation of electrical activity in excitable cells generating of electrical activity Nature. 2003; Scopus Google Scholar, E. Nature. 2002; PubMed Scopus Google Scholar). The expression of TRPM4 transcripts both in human and mouse tissues (Fig. is in of a functional role in excitable cells. Since the and dependence of TRPM4b is of Ca2+-activated channels we have taken to between these two currents in cells. TRPM4b could be from in the following currents through TRPM4b completely extracellular monovalent cations are by the is to in the the channel conductance is completely different for TRPM4b for B. Droogmans G. Physiol. 2003; PubMed Scopus Google and rectification of TRPM4b is not by [Ca2+]i B. Droogmans G. Physiol. 2003; PubMed Scopus Google Scholar). Ca2+ of TRPM4b to be identical at positive potential and negative potentials at between and 1 Ca2+ than cells for data not the dependence of channel was independent of [Ca2+]i (Fig. It is therefore that at negative and activation at positive potentials be by a low Ca2+ at negative and at positive potentials, as is the for Ca2+-activated channels B. Droogmans G. Physiol. 2003; PubMed Scopus Google Scholar, A. C. J. Gen. Physiol. 2000; PubMed Scopus Google Scholar). In addition, we have not in the rectification behavior at different which a voltage-dependent Ca2+ of At we the of the voltage-dependent of on our a block by intra- or extracellular divalent as for (4Voets T. Janssens A. Prenen J. Droogmans D. Nilius G. J. Gen. Physiol. 2003; 121: 245-260Crossref PubMed Scopus (137) Google be of voltage-gated channels on the voltage-dependent of residues in the fourth transmembrane domain with part of a residues are in and the fourth transmembrane domain of TRPM4, which could to the but are to this In we show for the first a Ca2+- and voltage-dependent TRP channel. features might be functionally in tissues that generate action potentials or modulate their potential a