Ca2+ Signaling by TRPC3 Involves Na+ Entry and Local Coupling to the Na+/Ca2+ Exchanger

Abstract

TRPC3 has been suggested as a key component of phospholipase C-dependent Ca2+ signaling. Here we investigated the role of TRPC3-mediated Na+ entry as a determinant of plasmalemmal Na+/Ca2+ exchange. Ca2+ signals generated by TRPC3 overexpression in HEK293 cells were found to be dependent on extracellular Na+, in that carbachol-stimulated Ca2+ entry into TRPC3 expressing cells was significantly suppressed when extracellular Na+ was reduced to 5 mm. Moreover, KB-R9743 (5 μm) an inhibitor of the Na+/Ca2+ exchanger (NCX) strongly suppressed TRPC3-mediated Ca2+ entry but not TRPC3-mediated Na+ currents. NCX1 immunoreactivity was detectable in HEK293 as well as in TRPC3-overexpressing HEK293 cells, and reduction of extracellular Na+ after Na+ loading with monensin resulted in significant rises in intracellular free Ca2+ (Ca2+i) of HEK293 cells. Similar rises in Ca2+i were recorded in TRPC3-overexpressing cells upon the reduction of extracellular Na+ subsequent to stimulation with carbachol. These increases in Ca2+i were associated with outward membrane currents at positive potentials and inhibited by KB-R7943 (5 μm), chelation of extracellular Ca2+, or dominant negative suppression of TRPC3 channel function. This suggests that Ca2+ entry into TRPC3-expressing cells involves reversed mode Na+/Ca2+ exchange. Cell fractionation experiments demonstrated co-localization of TRPC3 and NCX1 in low density membrane fractions, and co-immunoprecipitation experiments provided evidence for association of TRPC3 and NCX1. Glutathione S-transferase pull-down experiments revealed that NCX1 interacts with the cytosolic C terminus of TRPC3. We suggest functional and physical interaction of nonselective TRPC cation channels with NCX proteins as a novel principle of TRPC-mediated Ca2+ signaling. TRPC3 has been suggested as a key component of phospholipase C-dependent Ca2+ signaling. Here we investigated the role of TRPC3-mediated Na+ entry as a determinant of plasmalemmal Na+/Ca2+ exchange. Ca2+ signals generated by TRPC3 overexpression in HEK293 cells were found to be dependent on extracellular Na+, in that carbachol-stimulated Ca2+ entry into TRPC3 expressing cells was significantly suppressed when extracellular Na+ was reduced to 5 mm. Moreover, KB-R9743 (5 μm) an inhibitor of the Na+/Ca2+ exchanger (NCX) strongly suppressed TRPC3-mediated Ca2+ entry but not TRPC3-mediated Na+ currents. NCX1 immunoreactivity was detectable in HEK293 as well as in TRPC3-overexpressing HEK293 cells, and reduction of extracellular Na+ after Na+ loading with monensin resulted in significant rises in intracellular free Ca2+ (Ca2+i) of HEK293 cells. Similar rises in Ca2+i were recorded in TRPC3-overexpressing cells upon the reduction of extracellular Na+ subsequent to stimulation with carbachol. These increases in Ca2+i were associated with outward membrane currents at positive potentials and inhibited by KB-R7943 (5 μm), chelation of extracellular Ca2+, or dominant negative suppression of TRPC3 channel function. This suggests that Ca2+ entry into TRPC3-expressing cells involves reversed mode Na+/Ca2+ exchange. Cell fractionation experiments demonstrated co-localization of TRPC3 and NCX1 in low density membrane fractions, and co-immunoprecipitation experiments provided evidence for association of TRPC3 and NCX1. Glutathione S-transferase pull-down experiments revealed that NCX1 interacts with the cytosolic C terminus of TRPC3. We suggest functional and physical interaction of nonselective TRPC cation channels with NCX proteins as a novel principle of TRPC-mediated Ca2+ signaling. Members of the canonical TRP (transient receptor potential) subfamily (TRPC family) have recently been suggested as key components of phospholipase C-dependent Ca2+ signaling in mammalian cells (1Vennekens R. Voets T. Bindels R.J. Droogmans G. Nilius B. Cell Calcium. 2002; 31: 253-264Google Scholar). In particular, TRPC3 has been repeatedly shown to form nonselective cation channels that allow permeation of both Na+ and Ca2+ (2Hurst R.S. Zhu X. Boulay G. Birnbaumer L. Stefani E. FEBS Lett. 1998; 422: 333-338Google Scholar, 3Zitt C. Obukhov A.G. Strubing C. Zobel A. Kalkbrenner F. Luckhoff A. Schultz G. J. Cell Biol. 1997; 138: 1333-1341Google Scholar, 4Lintschinger B. Balzer-Geldsetzer M. Baskaran T. Graier W.F. Romanin C. Zhu M.X. Groschner K. J. Biol. Chem. 2000; 275: 27799-27805Google Scholar, 5Thyagarajan B. Poteser M. Romanin C. Kahr H. Zhu M.X. Groschner K. J. Biol. Chem. 2001; 276: 48149-48158Google Scholar). Stimulation of TRPC3 channels under physiological conditions gives rise to large Na+ inward currents, which cause substantial membrane depolarization and Na+ loading. The role of TRPC3 channels in cellular Ca2+ signaling is complex and incompletely understood. Recent evidence suggests that TRPC3 contributes significantly to Ca2+ entry by forming multiple types of Ca2+ entry channels including store-operated Ca2+ entry channels that are linked to the filling state of the endoplasmic reticulum (5Thyagarajan B. Poteser M. Romanin C. Kahr H. Zhu M.X. Groschner K. J. Biol. Chem. 2001; 276: 48149-48158Google Scholar, 6Trebak M. Bird G.S. McKay R.R. Putney Jr., J.W. J. Biol. Chem. 2002; 277: 21617-21623Google Scholar). Nonetheless, the most prominent electrophysiological phenomenon observed in TRPC3 expressing cells is a depolarizing Na+ inward current that has so far largely been neglected in terms of functional consequences. A Ca2+ transport system that is sensitive to changes in both intracellular Na+ and membrane potential is the plasma membrane Na+/Ca2+ exchanger (NCX). 1The abbreviations used are: NCX, Na+/Ca2+ exchanger; HEK, human embryonic kidney; PBS, phosphate-buffered saline; KB-R7943, (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate); GST, glutathione S-transferase; CCh, carbachol; HA, hemagglutinin; MES, 2-(N-morpholino)ethanesulfonic acid. This system is well known to contribute to efficient Ca2+ extrusion during activation of cardiac muscle, neurons, and endocrine cells and may contribute to Ca2+ entry as well because of operation in reversed mode (7Sham J.S. Cleemann L. Morad M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 121-125Google Scholar, 8Sage S.O. van Breemen C. Cannell M.B. J. Physiol. 1991; 440: 569-580Google Scholar, 9Crespo L.M. Grantham C.J. Cannell M.B. Nature. 1990; 345: 618-621Google Scholar, 10Van Eylen F. Lebeau C. Albuquerque-Silva J. Herchuelz A. Diabetes. 1998; 47: 1873-1880Google Scholar). Na+ entry and membrane depolarization as a typical result of TRPC3 activation is expected to exert substantial impact on NCX, leading to either reduced Ca2+ extrusion or to Ca2+ entry upon shift of the exchanger to reversed mode. Notably, both TRPC3 and NCX proteins appear to be tightly linked to Ca2+ homeostasis of the endoplasmic reticulum and mitochondria and may thus both be targeted to a domain of the plasma membrane that communicates with these intracellular Ca2+ stores (5Thyagarajan B. Poteser M. Romanin C. Kahr H. Zhu M.X. Groschner K. J. Biol. Chem. 2001; 276: 48149-48158Google Scholar, 11Zhang Z. Tang J. Tikunova S. Johnson J.D. Chen Z. Qin N. Dietrich A. Stefani E. Birnbaumer L. Zhu M.X. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3168-3173Google Scholar, 12Brini M. Manni S. Carafoli E. J. Biol. Chem. 2002; 277: 38693-38699Google Scholar). Here we tested the hypothesis of cross-signaling between TRPC3 channels and NCX in HEK293 cells, a cell type that has been extensively used as an expression system for the characterization of TRPC3 function. We present evidence for physical and functional coupling of TRPC3 and NCX1 and suggest TRPC3-mediated Na+ entry and Na+ accumulation at the cytoplasmic face of TRPC-associated NCX proteins as a key mechanism by which TRPC3 governs global and local Ca2+ signaling. Cell Culture and Transfections—HEK293 cells stably transfected with the coding region of HA-tagged TRPC3 (accession number U47050; designated as T3-9), with an N-terminal YFP fusion of TRPC3 (YFP-TRPC3; designated as YT3-2; TRPC3 was subcloned using BamHI/XbaI into the pEYFP-C1 vector (Clontech)), or with pEYFP only (designated as HEK-VT; controls) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 0.178 g/liter Geneticin (G418 sulfate). In case of control cells, different clones were pooled together and cultured as specified above. Some experiments were performed with wild type HEK293 cells that were cultured in the absence of G418 and transiently transfected by electroporation with either YFP-TRPC3 or pEYFP (controls). Measurement of Intracellular Ca2+ and Membrane Currents—The cells were loaded with fura-2/pentaacetoxymethyl ester (Molecular Probes, Eugene, OR) for 60 min at 37 °C and 5% CO2. Thereafter, the cells were washed and constantly perfused during experiments with buffers containing either 137 mm NaCl, 5.4 mm KCl, 10 mm HEPES (nominally Ca2+ free); 5 mm NaCl, 150 mm N-methylglucamine, 5.4 mm KCl, 10 mm HEPES (nominally Ca2+ free); or 5 mm NaCl, 150 mm N-methylglucamine, 5.4 mm KCl, 10 mm HEPES, 2 mm CaCl2. The free Ca2+ concentration of this nominally Ca2+-free solution was determined using a Ca2+-sensitive electrode and was ∼20 μm (15–30 μm). Excitation light was supplied via a Polychrome II polychromator (TILL Photonics, Gräfelfing, Germany), and emission was detected by a Sensicam CCD camera (PCO Computer Optics, Kelheim, Germany). Ca2+-sensitive fura-2 fluorescence was measured ratiometrically at 340- and 380-nm excitation wavelengths, and emission was collected at 510 nm. The digital image recordings were analyzed using Axon Imaging Work-bench (Axon Instruments, Foster City, CA). For simultaneous measurements of membrane conductances and intracellular free Ca2+, the membrane currents were recorded in fura-2-loaded cells with the perforated patch technique (13Poteser M. Wakabayashi I. Rosker C. Teubl M. Schindl R. Soldatov Romanin C. Groschner K. 92: using in at a concentration of in the were a of and were with a solution containing mm mm 10 mm HEPES, 5 mm mm with of with The currents were recorded at using an patch (Axon Instruments, Foster City, CA). The signals were low at and with 5 to potential were by (Axon Instruments, Foster City, CA). of the experiments were performed at of Cell cells were washed with PBS, the in 5 of PBS, at and in of The cells were by and of in mm MES, NaCl, was the was in a by of and of 5% in containing and for at The fractions, at the of the were collected to a of the was collected as and were collected as The proteins in the were with of for min on the proteins were in of for and cells were washed with PBS, the in 5 of and at The were in of mm 150 mm NaCl, and inhibitor and for min on The cell were and at for at were by of with of for at were with either 10 of or with 10 of at of were to the complex for 2 at was performed in the absence of only to the The were washed with in of and at °C for 5 The cell and were by and to were for at with mm 137 mm NaCl, mm KCl, containing and with either NCX or at with and NCX and were used as The were using an system Glutathione N-terminal as well as a of TRPC3 were into the fusion vector The were into which were in of medium containing at 37 of the fusion as and was by mm The cells were by and in containing a of cells were on and by to a concentration of The cell were at for min at The was and the was in PBS, and as above. The were pooled and with of a of for min at The were at for 5 min at °C and washed with of was and the proteins were with mm reduced analyzed on with type HEK293 cells or cells were as for pull-down experiments were performed by 10 of HEK293 cell of with either with or the or fusion in a of of for 2 at The were washed with The proteins were with of at °C for 5 and analyzed by and The were with at and using the were the medium was fura-2/pentaacetoxymethyl ester was The was and were were as the The were significant at using for TRPC3-mediated Ca2+ into HEK293 and by or overexpression of TRPC3 in cells has repeatedly been shown to result in Ca2+ which is in Ca2+ typical intracellular Ca2+ signals in the stably TRPC3 expressing cell The cells were with μm) in nominally Ca2+-free and extracellular Ca2+ was to 2 mm to Ca2+ Ca2+ signals were suppressed in the of 5 μm KB-R7943, an inhibitor of Na+/Ca2+ with for the reversed and significantly when experiments were performed at low extracellular Na+ (5 Similar were with cells stably expressing YFP-TRPC3 Ca2+ entry into wild type HEK293 cells or was of the extracellular Na+ concentration Na+ a Na+/Ca2+ in HEK293 We that TRPC3-mediated Na+ loading and membrane depolarization may Na+/Ca2+ in HEK293 cells, leading to either Ca2+ extrusion or Ca2+ entry via reversed mode exchange. Na+ entry and membrane depolarization during TRPC3 activation is expected to be at reduced extracellular the that reduction of extracellular Na+ is an that increases the for reversed mode NCX at a of intracellular Na+, this is expected to Ca2+ entry via NCX when Na+ loading and membrane depolarization are the key for of reversed mode NCX, as for stimulation of TRPC3 This the observed suppression of TRPC3-mediated Ca2+ signaling at low extracellular this hypothesis we for the expression of functional NCX in HEK293 cells. that an an NCX1 typical of proteins J.S. Chen F. A. E. N. Acad. Sci. Scholar, M. Groschner K. B. K. J. Biol. Chem. of a prominent at as well as a of in the of to the expected of NCX1. The a form of which may either a or a form of NCX1 Eylen F. A. Herchuelz A. Cell Calcium. 2001; Scholar). NCX in HEK293 cells was demonstrated in experiments in which cellular Na+ were to Ca2+ entry via reversed mode of extracellular Na+ to cellular Na+ loading a substantial shift in the for Na+/Ca2+ for operation of NCX in reversed mode. that a reduction of extracellular Na+ to 5 mm after Na+ loading of the cells with monensin μm) in a significant of cytoplasmic Ca2+ This of Ca2+i was dependent on extracellular Ca2+, inhibited by 5 μm KB-R7943 and observed after stimulation of phospholipase C with Na+ rises in Ca2+i of Na+ loaded cells were inhibited by KB-R7943 (5 μm). HEK293 cells a Ca2+ entry mechanism that is dependent on the Na+ with NCX operation in reversed mode. the hypothesis that Na+ entry TRPC3 channels is linked to Ca2+ homeostasis via of reversed mode NCX, we performed experiments in which TRPC3 channels were in nominally to allow TRPC3-mediated Na+ by reduction of extracellular that reduction of extracellular Na+ to 5 mm subsequent to stimulation with at extracellular Na+ is to cause significant rises in which are inhibited by KB-R7943 (5 in Ca2+i by subsequent of extracellular Ca2+ were significantly in the of KB-R7943 as with Ca2+i signals recorded during Na+ reduction were dependent on extracellular Ca2+, because of extracellular Ca2+ ∼20 μm (nominally to a μm by of mm the Na+ increases in Ca2+i of extracellular Na+ to 5 mm rises in Ca2+i of cells as well as in HEK293 cells that YFP-TRPC3 transiently of TRPC3 with a dominant negative of TRPC3 that is known to channel K. S. B. M. Romanin C. Zhu X. FEBS Lett. 1998; the Ca2+ signals by reduction of extracellular Na+ TRPC3 expression a Ca2+ entry in HEK293 cells. We suggest that TRPC3-mediated Ca2+ entry is in the of Na+ entry that is to intracellular Ca2+ signals by a via reversed mode in HEK293 cells. of Ca2+-sensitive fura-2 fluorescence recorded using a Na+ The cells were in nominally Ca2+-free solution containing 137 mm and after with μm CCh, at was reduced to 5 and 2 mm Ca2+ was after The the in the absence and of KB-R7943 Na+ increases in intracellular Ca2+ in different cell vector transfected cells stably expressing and transiently expressing YFP-TRPC3 The of the Na+ reduction changes in intracellular Ca2+ are in for nominally Ca2+ free a solution containing mm as well as in cells transfected to The significant the in cells under control Ca2+ by of Na+ with a the Ca2+ entry we performed simultaneous measurements of Ca2+i and membrane currents with the perforated patch technique in cells as well as in cells transiently transfected to a YFP-TRPC3 fusion typical recordings in the absence and of 5 μm of membrane as increases in membrane currents measured at Similar were observed in a of experiments using as low as μm an component of membrane was detectable during of Ca2+ intracellular stores The cell to reduction of extracellular Na+ were an Ca2+i and a in membrane The changes in membrane currents were of an of inward currents by a in outward currents at positive potentials A of the current to recorded during stimulation and Na+ reduction is in outward currents were recorded at depolarization during reduction of extracellular Na+ The current to under these conditions was different that of the nonselective of TRPC3-expressing cells and which for large inward currents by Na+ The typical inward currents at were not by 5 μm KB-R7943 and of KB-R7943 to Na+ inward currents in cells not that TRPC3 channels are not a of KB-R7943 the outward currents by Na+ a of reversed mode Na+/Ca2+ as well as the associated increases in Ca2+i were by KB-R7943 a of current measured at in cells during reduction of extracellular Na+ in the absence and the of 5 μm Na+ outward currents were not detectable in wild type HEK293 or vector with the observed of Na+ Ca2+ signals expression of TRPC3 a Ca2+ entry that is dependent on extracellular Na+, sensitive to the NCX inhibitor KB-R7943 and associated with outward currents at positive These strongly suggest a functional coupling between NCX and TRPC3 via cellular Na+ reduction a outward current in carbachol-stimulated of currents measured at in cells during reduction of extracellular Na+ to 5 mm in the absence and of KB-R7943 increases in current density measured at in and in cells with 5 μm KB-R7943 The a significant the in cells under control TRPC3 and NCX1 in of HEK293 TRPC3 channels are targeted to of the plasma T. X. J. Biol. Chem. 2001; 276: which be on low we performed cell fractionation experiments using as a for a co-localization of TRPC3 and NCX in TRPC3 immunoreactivity in terms of the was detected in the low density membrane and These low density in NCX1. is of that NCX1 immunoreactivity was detected in density fractions, to intracellular These the of a between TRPC3 and NCX1 in the plasma membrane and to for a between these transport in terms of by co-immunoprecipitation shown in significant of TRPC3 were using an NCX and of with an the of the TRPC3 in cells NCX1 immunoreactivity as shown for the a of cellular NCX1 is suggested to with TRPC3 to form a Ca2+ signaling and were with or and to or NCX, proteins cell proteins with the These are of NCX1 to the C of the physical interaction between NCX1 and we performed pull-down experiments using the cytosolic and of the TRPC3 as shown in NCX1 in terms of both NCX1 typical and was HEK293 by the C terminus but not by the terminus or in control the C terminus of TRPC3 is to with association of the transport in HEK293 cells. evidence for a novel type of Ca2+ signaling complex that is of the Na+ and Ca2+ proteins TRPC3 and NCX1. We a functional coupling between the transport on both local and physical association a signaling TRPC3 Ca2+ in by of a Na+ has been demonstrated to Ca2+ entry and to form nonselective cation channels that for Na+ and Ca2+ M. S. U. A. L. J. Droogmans G. Nilius B. J. Physiol. Scholar). large Na+ inward currents are measured during stimulation of phospholipase in cells TRPC3 (2Hurst R.S. Zhu X. Boulay G. Birnbaumer L. Stefani E. FEBS Lett. 1998; 422: 333-338Google Scholar, 3Zitt C. Obukhov A.G. Strubing C. Zobel A. Kalkbrenner F. Luckhoff A. Schultz G. J. Cell Biol. 1997; 138: 1333-1341Google Scholar, 4Lintschinger B. Balzer-Geldsetzer M. Baskaran T. Graier W.F. Romanin C. Zhu M.X. Groschner K. J. Biol. Chem. 2000; 275: 27799-27805Google Scholar, M. S. U. A. L. J. Droogmans G. Nilius B. J. Physiol. Scholar). to a complex between the TRPC-mediated cation and Ca2+ homeostasis Na+ entry and reversed mode Na+/Ca2+ which is associated with outward membrane currents at positive In a Ca2+ of Na+ transport may be Na+ entry is expected to during activation of TRPC3 channels in Ca2+ free Ca2+ entry into the cells via reversed mode Na+/Ca2+ may during Ca2+ The of the mechanism that this phenomenon at physiological extracellular Na+ as as the local Na+ concentration at the cytoplasmic face of the exchanger a during depolarization of the cells. TRPC3-mediated Na+ entry is to shift the potential of Na+/Ca2+ to negative the membrane in reversed mode NCX that local Na+ to at with physiological extracellular Na+ and Ca2+ mm Na+ and 2 mm and a cytoplasmic Ca2+ of is This may well the in TRPC3-expressing HEK293 cells that membrane potentials to The that local Na+ rises to mm during TRPC3 activation is a low in of the intracellular Na+ that have been for TRPC3-expressing as M. R. N. S. J. Physiol. Scholar, W.F. J. E. M. J. Physiol. 1998; Scholar). phospholipase C cells expressing TRPC3 are to complex changes in membrane potential as the potential of cells that may well positive the expected K. Graier W.F. Scholar, J. M. M. Graier W.F. J. Physiol. 2000; Scholar). reversed mode NCX by TRPC3-mediated Na+ loading and membrane depolarization is suggested as a potential mechanism of Ca2+ entry into cells expressing both TRPC3 and NCX The physiological of this to be NCX1 TRPC3-mediated Na+ and Membrane into Ca2+ on experiments HEK293 cells as an expression system for NCX1 that expression of NCX1 is H. J. J. N. Acad. Sci. 2002; Scholar, Cleemann L. Soldatov Morad M. J. Physiol. we present evidence for expression of NCX1 in HEK293 cells. experiments with HEK293 revealed a of proteins that is observed in NCX expressing cells, including a at and the immunoreactivity in pull-down experiments with a TRPC3 as a of and to and of NCX1 in cell types Eylen F. A. Herchuelz A. Cell Calcium. 2001; Scholar, L. Carafoli E. J. Biol. Chem. 2000; 275: Scholar, J. Physiol. 1998; 275: Scholar). Moreover, we evidence for a Ca2+ transport system in HEK293 cells. This system is in that Ca2+ entry is associated with outward membrane currents and sensitive to by the NCX inhibitor KB-R7943 at that are for of reversed mode NCX 2001; Scholar). and functional experiments evidence for expression of a functional NCX in HEK293 cells and suggest this as a signaling of TRPC3. The of a of NCX1 to the typical Ca2+ signals was of the signals to by KB-R7943 T. T. M. J. Biol. Chem. Scholar). We observed that only TRPC3 cells rises in intracellular Ca2+ upon reduction of extracellular Na+ to 5 that TRPC3-mediated Na+ loading and membrane depolarization is to shift the of Na+/Ca2+ to Ca2+ entry via reversed mode exchange. In with this the rises in intracellular Ca2+ by Na+ reduction were by 5 μm KB-R7943 and when extracellular Ca2+ was μm) by Moreover, expression of a dominant negative of which is known to TRPC3 the Ca2+ signals by reduction of extracellular Na+, the that these Ca2+ signals both functional TRPC3 and measurements of intracellular Ca2+ and membrane conductances demonstrated that the Na+ rises in Ca2+i of TRPC3-expressing cells were associated with membrane currents that reversed at and were sensitive to by of the current to of this phenomenon revealed large outward currents at potentials positive that as expected for reversed mode Na+/Ca2+ exchange. we observed different types of membrane currents in TRPC3-expressing HEK293 that to the nonselective TRPC3 and a that of Na+/Ca2+ exchange. KB-R7943 not the typical TRPC3-mediated inward Na+ currents by phospholipase C that of Ca2+ signaling is not to on TRPC3 cation channels but of reversed mode Na+/Ca2+ exchange. These strongly suggest that NCX1 is to local of intracellular Na+ that are associated with of TRPC3 channels and Na+ into a cellular Ca2+ NCX1 and TRPC3 a Ca2+ both NCX and to membrane has been T. X. J. Biol. Chem. 2001; 276: Scholar, J. M. N. Acad. Sci. 2002; Scholar). In with the of functional we that the cation transport co-localization in membrane of HEK293 cells. NCX1 immunoreactivity was detected in TRPC3 low density membrane Moreover, co-immunoprecipitation experiments provided evidence for association of NCX1 with TRPC3 in TRPC3 as well as NCX1 have been to with proteins of is to a of the signaling complex to as a key of a complex is because NCX1 has been to with but not J. M. N. Acad. Sci. 2002; Scholar). pull-down experiments that NCX1 is to with the of TRPC3. we of proteins experiments suggest a and functional between a TRP channel and NCX1 a signaling to be NCX1 as a signaling for nonselective channels of the TRP This signaling is suggested to Ca2+ signaling as well as of the plasma membrane with intracellular Ca2+ stores as the endoplasmic reticulum and mitochondria as has been suggested for both NCX1 M. Manni S. Carafoli E. J. Biol. Chem. 2002; 277: 38693-38699Google and TRPC3 K. Zhu M.X. S. Scholar, G. Bird Putney Jr., J.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: in terms of signaling the physical coupling of the transport to that low of TRPC3-mediated Na+ currents may be to a local Na+ that is to an intracellular Ca2+ by the associated NCX as in the in is of that membrane potential is a key determinant of this in that depolarization Ca2+ entry via reversed mode Na+/Ca2+ In of the of TRPC3 to a of signals as phospholipase intracellular Ca2+ S.O. van Breemen C. Cannell M.B. J. Physiol. 1991; 440: 569-580Google and M. B. Groschner K. into local Na+ entry and membrane association with NCX1 of physiological and in a of In we present evidence for the of a type of signaling of a nonselective TRPC channel and a NCX are to an efficient system for by activation of nonselective cation channels to a of local and global Ca2+ The of this novel may cell types and on membrane potential and local Na+ The signaling between a nonselective cation channel and an may a that is in the of Ca2+ homeostasis by nonselective cation We Kahr for We and for