Deciphering the Molecular Basis for the Pacemaker Signal

Abstract

Ask a competent predoctoral or medical student about the ionic basis of pacing in the mammalian heart and you will obtain a reasonable erudite reply, detailing the various ion channels that contribute to the pacemaker signal. Ask a similar question about the pacing system in the gastrointestinal tract and expect silence. Although work during the 1990s has unequivocally shown that the pacemaker signal to gastrointestinal smooth muscle originates from interstitial cells of Cajal1Huizinga J.D. Thuneberg L. Kluppel M. Malysz J. Mikkelsen H.B. Bernstein A. W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity.Nature. 1995; 373: 347-349Google Scholar, 2Ward S.M. Burns A.J. Torihashi S. Sanders K.M. Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.J Physiol. 1994; 480: 91-97Google Scholar the definitive membrane pathways that lead to cyclic oscillations in membrane voltage (the slow wave) in interstitial cells of Cajal and, subsequently, in smooth muscle remain elusive.Why is this important? Gastrointestinal contractions only occur when the membrane potential of smooth muscle cells is depolarized to a threshold to activate L-type Ca2+ channels and increase calcium influx into the smooth muscle cells.3Farrugia G. Ionic conductances in gastrointestinal smooth muscles and interstitial cells of Cajal.Ann Rev Physiol. 1999; 61: 45-84Google Scholar Therefore, the properties of the slow wave determine both chronotropicity (number of slow waves per minute, setting the maximum contractile frequency) and ionotropicity (level of depolarization determining the strength of contraction). Given that the slow wave originates from interstitial cells of Cajal, it is not surprising that the mechanism of pacemaker signal generation in interstitial cells of Cajal that initiates the slow wave has attracted the attention of researchers, pharmaceutical companies, clinicians, and patients.In an elegant study in this issue of Gastroenterology, Kim et al4Kim B.J. Lim H.-H. Ki Yang D. Jun J.Y. Chang I.Y. Park C.S. So I. Stanfield P.R. Kim K.W. Melastatin-type transient receptor potential channel 7 is required for intestinal pacemaking activity.Gastroenterology. 2005; 129: 1504-1517Abstract Full Text Full Text PDF Scopus (113) Google Scholar provide strong evidence that, in mice, the TRPM7 ion channel is required for intestinal pacing and that treatment of cell cultures containing interstitial cells of Cajal with TRPM7-specific RNA interference results in loss of pacemaker activity. For most of us, this leads to two immediate questions: what is TRPM7 and can we now definitively attribute the pacing signal in interstitial cells of Cajal to TRPM7?The protein that gives rise to the first described transient receptor potential (TRP) channel was discovered in Drosophila in 19695Cosens D.J. Manning A. Abnormal electroretinogram from a Drosophila mutant.Nature. 1969; 224: 285-287Google Scholar and the trp gene was cloned in 1989.6Montell C. Rubin G.M. Molecular characterization of the Drosophila trp locus a putative integral membrane protein required for phototransduction.Neuron. 1989; 2: 1313-1323Google Scholar The trp gene encodes a Ca2+ -permeable cation channel. A consensus report in 2002 proposed a now widely used unified nomenclature for the TRP superfamily.7Montell 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. A unified nomenclature for the superfamily of TRP cation channels.Mol Cell. 2002; 9: 229-231Google Scholar It classified TRP channels into TRPC (for canonical TRP), TRPV (named after the first member of the subfamily, the vanilloid receptor), and TRPM (also named after the first member, melastatin). Four further subfamilies have since been added to the classification, TRPA (ankyrin repeats), TRPML (mucolipins), TRPN (no mechanoreceptor potential C gene), and TRPP (polycystins).8Clapham D.E. Montell C. Schultz G. Julius D. International Union of Pharmacology. XLIII. Compendium of voltage-gated ion channels: transient receptor potential channels.Pharmacol Rev. 2003; 55: 591-596Google Scholar, 9Montell C. The TRP superfamily of cation channels.Science’s Signal Transduction Knowledge Environment. 2005; 272: re3Google Scholar Unlike most other ion channel families, the TRP superfamily of ion channels is identified based only on homology and not function, with homology generally limited to the transmembrane segments and a 25 amino acid motif, the TRP domain, which contains the TRP box (EWKFAR). The function of the different ion channels that make up this superfamily is diverse, with a common theme being a sensory function.10Padinjat R. Andrews S. TRP channels at a glance.J Cell Sci. 2004; 117: 5707-5709Google Scholar, 11Huang C.L. The transient receptor potential superfamily of ion channels.J Am Soc Nephrol. 2004; 15: 1690-1699Google Scholar All are cation channels with 6 transmembrane segments. They lack the voltage-sensing positively charged amino acids in the fourth transmembrane segment and are therefore relatively voltage insensitive. TRP channels are nonselective with PCa/PNa ≤10. The exceptions are TRPM4 and TRPM5 (monovalent-selective) and TRPV5 and TRPV6 (Ca2+-selective, PCa/PNa > 100).Can the pacing signal be definitively attributed to TRPM7? Our current understanding of how the pacemaker current is generated in interstitial cells of Cajal breaks down to mechanisms that appear to be incompatible. The proposed mechanism that has the largest body of evidence is that a nonselective cation channel is responsible for the pacing depolarizations. The published data in support of this hypothesis suggest that the nonselective cation channel is inhibited by calcium, and when submembrane intracytoplasmic calcium levels decrease, the channel opens, resulting in entry of positive ions and membrane depolarization.12Koh S.D. Ward S.M. Ordog T. Sanders K.M. Horowitz B. Conductances responsible for slow wave generation and propagation in interstitial cells of Cajal.Curr Opin Pharmacol. 2003; 3: 579-582Google Scholar, 13Sanders K.M. Koh S.D. Ordog T. Ward S.M. Ionic conductances involved in generation and propagation of electrical slow waves in phasic gastrointestinal muscles.Neurogastroenterol Motil. 2004; 16: 100-105Google Scholar The other major proposed mechanism is that a chloride channel is responsible for the rhythmic depolarizations that lead to the upstroke of the slow wave.14Huizinga J.D. Zhu Y. Ye J. Molleman A. High-conductance chloride channels generate pacemaker currents in interstitial cells of Cajal.Gastroenterology. 2002; 123: 1627-1636Google Scholar, 15Zhu Y. Mucci A. Huizinga J.D. Inwardly rectifying chloride channel activity in intestinal pacemaker cells.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G809-G821Google Scholar Another alternative mechanism proposed is that ether a-go-go-related gene (ERG) potassium channels may function as the pacemaker channel.16Zhu Y. Golden C.M. Ye J. Wang X.Y. Akbarali H.I. Huizinga J.D. ERG K+ currents regulate pacemaker activity in ICC.Am J Physiol Gastrointest Liver Physiol. 2003; 285: G1249-G1258Google Scholar The known properties of TRPM7 appear to be at odds with these hypotheses. Does this exclude TRPM7 as a candidate? Other TRP channels have been proposed as candidates for the nonselective cation channel, such as TRPC4.17Torihashi S. Fujimoto T. Trost C. Nakayama S. Calcium oscillation linked to pacemaking of interstitial cells of Cajal requirement of calcium influx and localization of TRP4 in caveolae.J Biol Chem. 2002; 277: 19191-19197Google Scholar The properties of all proposed channels to date are also not fully compatible with the interstitial cells of Cajal pacemaker. Therefore, given that there are opposing views, it is not unreasonable to advance yet another, providing that one has the data to make the argument. In this study, the authors do just that. In a meticulous study, they show that the cation selectivity of the pacemaker current in cultured murine interstitial cells of Cajal was similar to that of TRPM7, that cultured interstitial cells of Cajal contained TRPM7 messenger RNA, that cultured and noncultured murine small intestinal interstitial cells of Cajal expressed the protein, that RNA interference targeting TRPM7 abolished the pacemaker current, and that TRPM7 was expressed in interstitial cells of Cajal but not smooth muscle cells.So, is the answer a definitive yes? As is so often the case, no. If one looks at all of the published data available on TRPM7, it is apparent that TRPM7 is ubiquitously expressed, that it is constitutively active, that its primary role is in cell viability and magnesium homeostasis, and that it passes little inward current under physiologic conditions.11Huang C.L. The transient receptor potential superfamily of ion channels.J Am Soc Nephrol. 2004; 15: 1690-1699Google Scholar Therefore, if one were looking for a pacemaker channel, one would not choose this channel as a prime candidate because of its ubiquitous distribution and because its roles to date do not coincide with the requirements of a pacemaker channel. These properties do not preclude an additional role for TRPM7 but require reconciliation between what is known about TRPM7 and the specialized expression and function of a channel that generates a pacemaker signal. Even if TRPM7 is found to be the predominant channel that generates the murine interstitial cell of Cajal pacemaker signal, it does not necessarily mean that the same applies across species, including humans. There are species differences that exist in ion channels expressed in interstitial cells of Cajal and, therefore, the presence of TRPM7 in murine interstitial cells of Cajal may not necessarily translate to other species. It is also important to keep in mind that loss of TRPM7 is known to affect cell viability.18Nadler 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. LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability.Nature. 2001; 411: 590-595Google Scholar Therefore, the RNA interference experiments showing loss of the pacemaker current, although suggestive of a role for TRPM7 as a pacemaker generator, can also be interpreted as a general loss of cell viability and not due to a direct effect on the pacemaker current.Where does this leave us? We are certainly farther along than before. We now have another attractive candidate for the pacemaker channel that will require extensive confirmation and expansion of the studies to other species. It is essential to determine whether TRPM7 is expressed in all interstitial cells of Cajal or only in the interstitial cells of Cajal that participate in generating the pacemaker signal, because different classes of interstitial cells of Cajal have different roles. TRPM7 has an atypical C-terminal protein kinase domain that, among other functions, mediates the effects of cyclic adenosine monophosphate (cAMP) on channel activity.19Takezawa R. Schmitz C. Demeuse P. Scharenberg A.M. Penner R. Fleig A. Receptor-mediated regulation of the TRPM7 channel through its endogenous protein kinase domain.Proc Natl Acad Sci U S A. 2004; 101: 6009-6014Google Scholar If it is the pacemaker channel, we need to better understand the function of the protein kinase domain and to know with what TRPM7 associates and its regulation. We need to record ionic currents from noncultured pacemaker interstitial cells of Cajal and perform the required selectivity experiments, including effect of intracellular magnesium. We also need to know if the pacemaker channel is the same in each region of the gastrointestinal tract. What we do know is that TRP channels can form heteromultimers. For example, TRPM7 may form heteromultimers with TRPM6.20Chubanov V. Waldegger S. Mederos y Schnitzler M. Vitzthum H. Sassen M.C. Seyberth H.W. Konrad M. Gudermann T. Disruption of TRPM6/TRPM7 complex formation by a mutation in the TRPM6 gene causes hypomagnesemia with secondary hypocalcemia.Proc Natl Acad Sci U S A. 2004; 101: 2894-2899Google Scholar It is very possible that the inability to reconcile all properties of the pacemaker current with a particular expressed channel may be due to the fact that the pacemaker channel is a heteromultimer. Luckily, there exists sufficient interest in this field that these answers should be soon forth coming. Ask a competent predoctoral or medical student about the ionic basis of pacing in the mammalian heart and you will obtain a reasonable erudite reply, detailing the various ion channels that contribute to the pacemaker signal. Ask a similar question about the pacing system in the gastrointestinal tract and expect silence. Although work during the 1990s has unequivocally shown that the pacemaker signal to gastrointestinal smooth muscle originates from interstitial cells of Cajal1Huizinga J.D. Thuneberg L. Kluppel M. Malysz J. Mikkelsen H.B. Bernstein A. W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity.Nature. 1995; 373: 347-349Google Scholar, 2Ward S.M. Burns A.J. Torihashi S. Sanders K.M. Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.J Physiol. 1994; 480: 91-97Google Scholar the definitive membrane pathways that lead to cyclic oscillations in membrane voltage (the slow wave) in interstitial cells of Cajal and, subsequently, in smooth muscle remain elusive. Why is this important? Gastrointestinal contractions only occur when the membrane potential of smooth muscle cells is depolarized to a threshold to activate L-type Ca2+ channels and increase calcium influx into the smooth muscle cells.3Farrugia G. Ionic conductances in gastrointestinal smooth muscles and interstitial cells of Cajal.Ann Rev Physiol. 1999; 61: 45-84Google Scholar Therefore, the properties of the slow wave determine both chronotropicity (number of slow waves per minute, setting the maximum contractile frequency) and ionotropicity (level of depolarization determining the strength of contraction). Given that the slow wave originates from interstitial cells of Cajal, it is not surprising that the mechanism of pacemaker signal generation in interstitial cells of Cajal that initiates the slow wave has attracted the attention of researchers, pharmaceutical companies, clinicians, and patients. In an elegant study in this issue of Gastroenterology, Kim et al4Kim B.J. Lim H.-H. Ki Yang D. Jun J.Y. Chang I.Y. Park C.S. So I. Stanfield P.R. Kim K.W. Melastatin-type transient receptor potential channel 7 is required for intestinal pacemaking activity.Gastroenterology. 2005; 129: 1504-1517Abstract Full Text Full Text PDF Scopus (113) Google Scholar provide strong evidence that, in mice, the TRPM7 ion channel is required for intestinal pacing and that treatment of cell cultures containing interstitial cells of Cajal with TRPM7-specific RNA interference results in loss of pacemaker activity. For most of us, this leads to two immediate questions: what is TRPM7 and can we now definitively attribute the pacing signal in interstitial cells of Cajal to TRPM7? The protein that gives rise to the first described transient receptor potential (TRP) channel was discovered in Drosophila in 19695Cosens D.J. Manning A. Abnormal electroretinogram from a Drosophila mutant.Nature. 1969; 224: 285-287Google Scholar and the trp gene was cloned in 1989.6Montell C. Rubin G.M. Molecular characterization of the Drosophila trp locus a putative integral membrane protein required for phototransduction.Neuron. 1989; 2: 1313-1323Google Scholar The trp gene encodes a Ca2+ -permeable cation channel. A consensus report in 2002 proposed a now widely used unified nomenclature for the TRP superfamily.7Montell 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. A unified nomenclature for the superfamily of TRP cation channels.Mol Cell. 2002; 9: 229-231Google Scholar It classified TRP channels into TRPC (for canonical TRP), TRPV (named after the first member of the subfamily, the vanilloid receptor), and TRPM (also named after the first member, melastatin). Four further subfamilies have since been added to the classification, TRPA (ankyrin repeats), TRPML (mucolipins), TRPN (no mechanoreceptor potential C gene), and TRPP (polycystins).8Clapham D.E. Montell C. Schultz G. Julius D. International Union of Pharmacology. XLIII. Compendium of voltage-gated ion channels: transient receptor potential channels.Pharmacol Rev. 2003; 55: 591-596Google Scholar, 9Montell C. The TRP superfamily of cation channels.Science’s Signal Transduction Knowledge Environment. 2005; 272: re3Google Scholar Unlike most other ion channel families, the TRP superfamily of ion channels is identified based only on homology and not function, with homology generally limited to the transmembrane segments and a 25 amino acid motif, the TRP domain, which contains the TRP box (EWKFAR). The function of the different ion channels that make up this superfamily is diverse, with a common theme being a sensory function.10Padinjat R. Andrews S. TRP channels at a glance.J Cell Sci. 2004; 117: 5707-5709Google Scholar, 11Huang C.L. The transient receptor potential superfamily of ion channels.J Am Soc Nephrol. 2004; 15: 1690-1699Google Scholar All are cation channels with 6 transmembrane segments. They lack the voltage-sensing positively charged amino acids in the fourth transmembrane segment and are therefore relatively voltage insensitive. TRP channels are nonselective with PCa/PNa ≤10. The exceptions are TRPM4 and TRPM5 (monovalent-selective) and TRPV5 and TRPV6 (Ca2+-selective, PCa/PNa > 100). Can the pacing signal be definitively attributed to TRPM7? Our current understanding of how the pacemaker current is generated in interstitial cells of Cajal breaks down to mechanisms that appear to be incompatible. The proposed mechanism that has the largest body of evidence is that a nonselective cation channel is responsible for the pacing depolarizations. The published data in support of this hypothesis suggest that the nonselective cation channel is inhibited by calcium, and when submembrane intracytoplasmic calcium levels decrease, the channel opens, resulting in entry of positive ions and membrane depolarization.12Koh S.D. Ward S.M. Ordog T. Sanders K.M. Horowitz B. Conductances responsible for slow wave generation and propagation in interstitial cells of Cajal.Curr Opin Pharmacol. 2003; 3: 579-582Google Scholar, 13Sanders K.M. Koh S.D. Ordog T. Ward S.M. Ionic conductances involved in generation and propagation of electrical slow waves in phasic gastrointestinal muscles.Neurogastroenterol Motil. 2004; 16: 100-105Google Scholar The other major proposed mechanism is that a chloride channel is responsible for the rhythmic depolarizations that lead to the upstroke of the slow wave.14Huizinga J.D. Zhu Y. Ye J. Molleman A. High-conductance chloride channels generate pacemaker currents in interstitial cells of Cajal.Gastroenterology. 2002; 123: 1627-1636Google Scholar, 15Zhu Y. Mucci A. Huizinga J.D. Inwardly rectifying chloride channel activity in intestinal pacemaker cells.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G809-G821Google Scholar Another alternative mechanism proposed is that ether a-go-go-related gene (ERG) potassium channels may function as the pacemaker channel.16Zhu Y. Golden C.M. Ye J. Wang X.Y. Akbarali H.I. Huizinga J.D. ERG K+ currents regulate pacemaker activity in ICC.Am J Physiol Gastrointest Liver Physiol. 2003; 285: G1249-G1258Google Scholar The known properties of TRPM7 appear to be at odds with these hypotheses. Does this exclude TRPM7 as a candidate? Other TRP channels have been proposed as candidates for the nonselective cation channel, such as TRPC4.17Torihashi S. Fujimoto T. Trost C. Nakayama S. Calcium oscillation linked to pacemaking of interstitial cells of Cajal requirement of calcium influx and localization of TRP4 in caveolae.J Biol Chem. 2002; 277: 19191-19197Google Scholar The properties of all proposed channels to date are also not fully compatible with the interstitial cells of Cajal pacemaker. Therefore, given that there are opposing views, it is not unreasonable to advance yet another, providing that one has the data to make the argument. In this study, the authors do just that. In a meticulous study, they show that the cation selectivity of the pacemaker current in cultured murine interstitial cells of Cajal was similar to that of TRPM7, that cultured interstitial cells of Cajal contained TRPM7 messenger RNA, that cultured and noncultured murine small intestinal interstitial cells of Cajal expressed the protein, that RNA interference targeting TRPM7 abolished the pacemaker current, and that TRPM7 was expressed in interstitial cells of Cajal but not smooth muscle cells. So, is the answer a definitive yes? As is so often the case, no. If one looks at all of the published data available on TRPM7, it is apparent that TRPM7 is ubiquitously expressed, that it is constitutively active, that its primary role is in cell viability and magnesium homeostasis, and that it passes little inward current under physiologic conditions.11Huang C.L. The transient receptor potential superfamily of ion channels.J Am Soc Nephrol. 2004; 15: 1690-1699Google Scholar Therefore, if one were looking for a pacemaker channel, one would not choose this channel as a prime candidate because of its ubiquitous distribution and because its roles to date do not coincide with the requirements of a pacemaker channel. These properties do not preclude an additional role for TRPM7 but require reconciliation between what is known about TRPM7 and the specialized expression and function of a channel that generates a pacemaker signal. Even if TRPM7 is found to be the predominant channel that generates the murine interstitial cell of Cajal pacemaker signal, it does not necessarily mean that the same applies across species, including humans. There are species differences that exist in ion channels expressed in interstitial cells of Cajal and, therefore, the presence of TRPM7 in murine interstitial cells of Cajal may not necessarily translate to other species. It is also important to keep in mind that loss of TRPM7 is known to affect cell viability.18Nadler 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. LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability.Nature. 2001; 411: 590-595Google Scholar Therefore, the RNA interference experiments showing loss of the pacemaker current, although suggestive of a role for TRPM7 as a pacemaker generator, can also be interpreted as a general loss of cell viability and not due to a direct effect on the pacemaker current. Where does this leave us? We are certainly farther along than before. We now have another attractive candidate for the pacemaker channel that will require extensive confirmation and expansion of the studies to other species. It is essential to determine whether TRPM7 is expressed in all interstitial cells of Cajal or only in the interstitial cells of Cajal that participate in generating the pacemaker signal, because different classes of interstitial cells of Cajal have different roles. TRPM7 has an atypical C-terminal protein kinase domain that, among other functions, mediates the effects of cyclic adenosine monophosphate (cAMP) on channel activity.19Takezawa R. Schmitz C. Demeuse P. Scharenberg A.M. Penner R. Fleig A. Receptor-mediated regulation of the TRPM7 channel through its endogenous protein kinase domain.Proc Natl Acad Sci U S A. 2004; 101: 6009-6014Google Scholar If it is the pacemaker channel, we need to better understand the function of the protein kinase domain and to know with what TRPM7 associates and its regulation. We need to record ionic currents from noncultured pacemaker interstitial cells of Cajal and perform the required selectivity experiments, including effect of intracellular magnesium. We also need to know if the pacemaker channel is the same in each region of the gastrointestinal tract. What we do know is that TRP channels can form heteromultimers. For example, TRPM7 may form heteromultimers with TRPM6.20Chubanov V. Waldegger S. Mederos y Schnitzler M. Vitzthum H. Sassen M.C. Seyberth H.W. Konrad M. Gudermann T. Disruption of TRPM6/TRPM7 complex formation by a mutation in the TRPM6 gene causes hypomagnesemia with secondary hypocalcemia.Proc Natl Acad Sci U S A. 2004; 101: 2894-2899Google Scholar It is very possible that the inability to reconcile all properties of the pacemaker current with a particular expressed channel may be due to the fact that the pacemaker channel is a heteromultimer. Luckily, there exists sufficient interest in this field that these answers should be soon forth coming. Melastatin-Type Transient Receptor Potential Channel 7 Is Required for Intestinal Pacemaking ActivityGastroenterologyVol. 129Issue 5PreviewBackground & Aims: Interstitial cells of Cajal are pacemakers in the gastrointestinal tract, regulating rhythmicity by activating nonselective cation channels. In Caenorhabditis elegans, the melastatin-type transient receptor potential (TRPM) channel, especially TRPM7, was suggested as being involved in defecation rhythm. The aim here was to show that the nonselective cation channel in interstitial cells of Cajal in mouse small intestine has properties essentially identical to those of murine TRPM7, heterologously expressed in human embryonic kidney cells. Full-Text PDF