The Recombinant Human TRPV6 Channel Functions as Ca2+Sensor in Human Embryonic Kidney and Rat Basophilic Leukemia Cells
2002; Elsevier BV; Volume: 277; Issue: 39 Linguagem: Inglês
10.1074/jbc.m202822200
ISSN1083-351X
AutoresMatthias Bödding, Ulrich Wissenbach, Veit Flockerzi,
Tópico(s)Magnesium in Health and Disease
ResumoThe activation mechanism of the recently cloned human transient receptor potential vanilloid type 6 (TRPV6) channel, originally termed Ca2+ transporter-like protein and Ca2+ transporter type 1, was investigated in whole-cell patch-clamp experiments using transiently transfected human embryonic kidney and rat basophilic leukemia cells. The TRPV6-mediated currents are highly Ca2+-selective, show a strong inward rectification, and reverse at positive potentials, which is similar to store-operated Ca2+ entry in electrically nonexcitable cells. The gating of TRPV6 channels is strongly dependent on the cytosolic free Ca2+ concentration; lowering the intracellular free Ca2+ concentration results in Ca2+ influx, and current amplitude correlates with the intracellular EGTA or BAPTA concentration. This is also the case for TRPV6-mediated currents in the absence of extracellular divalent cations; compared with endogenous currents in nontransfected rat basophilic leukemia cells, these TRPV6-mediated monovalent currents reveal differences in reversal potential, inward rectification, and slope at very negative potentials. Release of stored Ca2+by inositol 1,4,5-trisphosphate and/or the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin appears not to be involved in TRPV6 channel gating in both cell lines but, in rat basophilic leukemia cells, readily activates the endogenous Ca2+ release-activated Ca2+ current. In conclusion, TRPV6, expressed in human embryonic kidney cells and in rat basophilic leukemia cells, functions as a Ca2+-sensing Ca2+ channel independently of procedures known to deplete Ca2+ stores. The activation mechanism of the recently cloned human transient receptor potential vanilloid type 6 (TRPV6) channel, originally termed Ca2+ transporter-like protein and Ca2+ transporter type 1, was investigated in whole-cell patch-clamp experiments using transiently transfected human embryonic kidney and rat basophilic leukemia cells. The TRPV6-mediated currents are highly Ca2+-selective, show a strong inward rectification, and reverse at positive potentials, which is similar to store-operated Ca2+ entry in electrically nonexcitable cells. The gating of TRPV6 channels is strongly dependent on the cytosolic free Ca2+ concentration; lowering the intracellular free Ca2+ concentration results in Ca2+ influx, and current amplitude correlates with the intracellular EGTA or BAPTA concentration. This is also the case for TRPV6-mediated currents in the absence of extracellular divalent cations; compared with endogenous currents in nontransfected rat basophilic leukemia cells, these TRPV6-mediated monovalent currents reveal differences in reversal potential, inward rectification, and slope at very negative potentials. Release of stored Ca2+by inositol 1,4,5-trisphosphate and/or the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin appears not to be involved in TRPV6 channel gating in both cell lines but, in rat basophilic leukemia cells, readily activates the endogenous Ca2+ release-activated Ca2+ current. In conclusion, TRPV6, expressed in human embryonic kidney cells and in rat basophilic leukemia cells, functions as a Ca2+-sensing Ca2+ channel independently of procedures known to deplete Ca2+ stores. intracellular free Ca2+ concentration Ca2+ transporter 1 Ca2+ transporter-like calmodulin Chinese hamster ovary ether-à-go-go intracellular EGTA concentration human embryonic kidney Ca2+release-activated Ca2+ current inositol 1,4,5-trisphosphate current-voltage Ca2+-activated K+ intracellular free Mg2+ concentration rat basophilic leukemia transient receptor potential transient receptor potential subfamily C (classical type) transient receptor potential subfamily M (melastatin type) transient receptor potential subfamily V (vanilloid-receptor type) wild-type Calcium is involved in a multitude of intracellular signal transduction mechanisms ranging from contraction to secretion. To achieve a high bandwidth of signal transmission with sometimes even opposing effects, cells need to control the spatio-temporal resolution of their Ca2+ signals (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4440) Google Scholar). In nonexcitable cells the rise in [Ca2+]i1during stimulation of G-protein-coupled receptors or receptor tyrosine kinases is regulated in a complex fashion by Ca2+ release from endogenous IP3-sensitive stores followed by store-operated Ca2+ influx across the plasma membrane. One of the best characterized store-operated Ca2+ entry pathways is the Ca2+ release-activated Ca2+current, termed ICRAC (2Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1291) Google Scholar, 3Putney Jr., J.W. Capacitative Calcium Entry. Springer, New York1997Crossref Google Scholar). The molecular identity of store-operated channels is a matter of debate. Several members of the TRP family, a group of Ca2+permeable channels related to the Drosophila melanogasterTRP gene product, have been implicated in store-dependent cation influx (4Clapham D.E. Runnels L.W. Strübing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (957) Google Scholar, 5Montell C. Birnbaumer L. Flockerzi V. Cell. 2002; 108: 595-598Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar). However, so far, none of the trp genes has been shown to encode channels with the ion-permeation properties and the sensitivity to store depleting agents of CRAC channels. The family of the trp genes in vertebrates can be divided into three subfamilies, based on similarities in the structures of the encoded proteins (6Montell C. Birnbaumer L. Flockerzi V. Bindels R.J. Bruford E.A. Caterina M.J. Clapham D.E. Harteneck C. Heller S. Julius D. Kojima I. Mori Y. Penner R. Prawitt D. Scharenberg A.M. Schultz G. Shimizu N. Zhu M.X. Mol. Cell. 2002; 9: 1-3Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar), the TRPC, the TRPM, and the TRPV subfamilies. Members of the TRPV subfamily appear to be regulated by physical or chemical stimuli such as heat, osmotic, or mechanical stress. The recently cloned human TRPV6 gene product (GenBankTMaccession no. CAC20417), formerly called CaT-L or TRP8 (7Wissenbach U. Niemeyer B.A. Fixemer T. Schneidewind A. Trost C. Cavalie A. Reus K. Meese E. Bonkhoff H. Flockerzi V. J. Biol. Chem. 2001; 276: 19461-19468Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 8Niemeyer B.A. Bergs C. Wissenbach U. Flockerzi V. Trost C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3600-3605Crossref PubMed Scopus (151) Google Scholar) or CaT1 (9Peng J.B. Chen X.Z. Berger U.V. Weremowicz S. Morton C.C. Vassilev P.M. Brown E.M. Hediger M.A. Biochem. Biophys. Res. Commun. 2000; 278: 326-332Crossref PubMed Scopus (188) Google Scholar), belongs into this group. Human TRPV6 channels (7Wissenbach U. Niemeyer B.A. Fixemer T. Schneidewind A. Trost C. Cavalie A. Reus K. Meese E. Bonkhoff H. Flockerzi V. J. Biol. Chem. 2001; 276: 19461-19468Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), like rat TRPV6 (formerly called rat CaT1; Ref. 10Peng J.B. Chen X.Z. Berger U.V. Vassilev P.M. Tsukaguchi H. Brown E.M. Hediger M.A. J. Biol. Chem. 1999; 274: 22739-22746Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar) and rabbit TRPV5 channels (former ECaC for epithelial Ca2+ channel; Ref. 11Vennekens 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), show a high Ca2+ selectivity, Ca2+ and Na+permeation properties indicative of an anomalous mole fraction behavior, and current-voltage relationships similar to CRAC channels. Recently these common features between TRPV6 and CRAC channels were demonstrated by two groups (12Yue L. Peng J.B. Hediger M.A. Clapham D.E. Nature. 2001; 410: 705-709Crossref PubMed Scopus (320) Google Scholar, 13Voets T. Prenen J. Fleig A. Vennekens R. Watanabe H. Hoenderop J.G. Bindels R.J. Droogmans G. Penner R. Nilius B. J. Biol. Chem. 2001; 276: 47767-47770Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). In addition Voets et al. (13Voets T. Prenen J. Fleig A. Vennekens R. Watanabe H. Hoenderop J.G. Bindels R.J. Droogmans G. Penner R. Nilius B. J. Biol. Chem. 2001; 276: 47767-47770Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) also revealed several differences between ICRAC in RBL cells and TRPV6-mediated Ca2+currents when expressed in HEK cells, including insensitivity of TRPV6 channels to store depletion and the effect of intracellular Mg2+, which causes voltage-dependent block of TRPV6, but not of CRAC channels. In contrast, Yue et al.(12Yue L. Peng J.B. Hediger M.A. Clapham D.E. Nature. 2001; 410: 705-709Crossref PubMed Scopus (320) Google Scholar) demonstrated activation of TRPV6 channels in CHO cells by depletion of calcium stores with IP3 and thapsigargin. This gating mechanism could be accomplished when recordings were performed 8–12 h but not 24 h after transfection (12Yue L. Peng J.B. Hediger M.A. Clapham D.E. Nature. 2001; 410: 705-709Crossref PubMed Scopus (320) Google Scholar). The authors assumed that, after 24 h, TRPV6 overexpression is not matched by the signal transduction apparatus presumed to be endogenously present in CHO cells and responsible for sensing store depletion. Subsequently, Yue et al. (12Yue L. Peng J.B. Hediger M.A. Clapham D.E. Nature. 2001; 410: 705-709Crossref PubMed Scopus (320) Google Scholar) suggested that the TRPV6 protein comprises all or a part of the CRAC pore. In the present study, two different cell lines, RBL and HEK cells, were used to study the gating of human TRPV6 channels. RBL cells are a common model system to study ICRAC, whereas in HEK cells ionic conductances associated with store depletion are not prominent (14Fasolato C. Nilius B. Pflügers Arch. 1998; 436: 69-74Crossref PubMed Scopus (96) Google Scholar, 15Fierro L. Lund P.E. Parekh A.B. Pflügers Arch. 2000; 440: 580-587PubMed Google Scholar, 16Mignen O. Shuttleworth T.J. J. Biol. Chem. 2000; 275: 9114-9119Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Accordingly, one could assume a higher or different expression level of the signal transduction machinery that leads to ICRAC activation, in RBL than in HEK cells. We therefore expressed the TRPV6 cDNA in both cells and studied whether there is a cell-specific modulation of TRPV6 channel function. Under conditions of high intracellular Ca2+ buffering, TRPV6-mediated Ca2+ currents develop to such an extent even in RBL cells that it swamps the current through CRAC channels. However, at low intracellular Ca2+ buffering, store depletion activates ICRAC but not TRPV6 channels, suggesting that the TRPV6 protein is not sensitive to store depletion under these conditions. Instead, TRPV6 channels function as Ca2+-sensing Ca2+ pores and their current amplitudes are inversely correlated with the [Ca2+]i. HEK-293 (ATCC, 1573-CRL) and RBL-1 cells (ATTC, 1378-CRL) were from the American Type Culture Collection (Manassas, VA). Minimum essential medium with Earle's salts andl-glutamine was used for HEK cells and Dulbecco's modified Eagle's medium including l-glutamine, sodium pyruvate, and 1000 mg/liter d-glucose for RBL cells. Both culture media were supplemented with 10% fetal calf serum (Invitrogen, Paisley, UK). Cell lines were grown in a 5% CO2 humidified incubator at 37 °C. Cells were plated onto glass coverslips in 35-mm diameter Petri dishes 24 h prior to transient transfection with 4 μg of DNA in 5 ml of the PolyFect® reagents (Qiagen, Hilden, Germany). The bicistronic expression plasmid pdiCaT-L was constructed as described (7Wissenbach U. Niemeyer B.A. Fixemer T. Schneidewind A. Trost C. Cavalie A. Reus K. Meese E. Bonkhoff H. Flockerzi V. J. Biol. Chem. 2001; 276: 19461-19468Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar) and contained the entire protein-coding regions of the b-variant of human TRPV6 (formerly CaT-Lb, DDBJ/EMBL/GenBankTM accession no. CAC20417) followed by an internal ribosomal entry side and the green fluorescence protein DNA. The a-variant of human TRPV6 (accession no. CAC20416) differs in three amino acid residues to that of the b-variant (R157C, V378M, and T681M) and is identical with the human TRPV6 variant (accession no. AAL40230) reported by Peng et al. (17Peng J.B. Brown E.M. Hediger M.A. Genomics. 2001; 76: 99-109Crossref PubMed Scopus (86) Google Scholar). For experiments, coverslips with cells were transferred 24–32 h after transfection to the recording chamber and kept in a modified Ringer's solution containing (in mm): 145 NaCl, 10 CaCl2, 10 CsCl, 2.8 KCl, 2 MgCl2, 11 glucose, 10 HEPES, adjusted to pH 7.2 with NaOH. The divalent-free solution contained (in mm): 145 NaCl, 2.8 KCl, 10 CsCl, 11 glucose, 10 EGTA, 10 HEPES, adjusted to pH 7.2 with NaOH. Patch-clamp experiments were conducted in the tight-seal whole-cell configuration (18Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflügers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15141) Google Scholar) using an EPC-9 amplifier (HEKA Elektronik, Lambrecht, Germany). Patch pipettes were pulled from borosilicate glass (Kimax®) and had resistances between 2 and 3 megohms when filled with the standard internal solution containing (in mm): 145 cesium glutamate, 10 HEPES, 8 NaCl, 1 MgCl2, 2 Mg-ATP adjusted to pH 7.2 with CsOH. When using high intracellular chelator concentrations (30 and 60 mm), the osmolarity was kept constant by appropriate reduction of cesium glutamate. The intracellular Mg-ATP concentration was increased to 6 mm for the experiments in the absence of external divalent cations. This was done to minimize contamination of monovalent currents presumably mediated by endogenously expressed TRPM7 channels in HEK and RBL cells (19Nadler 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 (800) Google Scholar). The Mg2+-free pipette solution contained 10 mm EGTA, Na2-ATP instead of the Mg2+ salt and no addition of MgCl2 to study the time-dependent removal of the intracellular Mg2+ block. The series resistances were typically in the range of 5–10 megohms and not electronically compensated. Currents were filtered using an 8-pole Bessel filter at 8 kHz and digitized at 100 μs. Voltage-clamp recordings were performed using ramps (−110 mV to +90 mV in 50 ms) applied every 2 s using PULSE software (HEKA Electronics) on a personal computer. Cells were held at −10 mV between ramps. Several parameters (capacitance, series resistance, holding current) were displayed simultaneously at a slower rate (2 Hz) using the X-Chart display (HEKA Electronics). The membrane potential values were corrected for 10-mV liquid junction potential. No additional voltage correction was performed for the experiments under divalent-free conditions. Effects of changes in surface charge screening were ignored. All experiments were carried out at room temperature (20–23 °C); internal solutions were kept on ice to minimize hydrolysis of the nucleotides. For Northern blot analysis, 2 μg of poly(A)+ RNA from rat duodenum and from RBL cells were separated by electrophoresis on 0.8% agarose gels and thereafter transferred to Hybond N nylon membranes (Amersham Biosciences Europe, Freiburg, Germany) as described (7Wissenbach U. Niemeyer B.A. Fixemer T. Schneidewind A. Trost C. Cavalie A. Reus K. Meese E. Bonkhoff H. Flockerzi V. J. Biol. Chem. 2001; 276: 19461-19468Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). The membranes were hybridized in the presence of 50% formamide at 42 °C overnight. No additional signals were detected if the exposure time was 1 week. The probe was the 1539-bp ApaI cDNA fragment of rat TRPV6 (DDBJ/EMBL/GenBankTM, accession no. AF160798) encoding amino acid residues 208–721 and the 345-bpEcoRI/BamHI fragment of human TRPV6 (amino acid residues 528–643), both labeled by random priming with [α-32P]dCTP. Analysis was performed with PulseFit and programs written in the IGOR macro language (Wave Metrics, Lake Oswego, OR). Ca2+ currents elicited by voltage ramps were leak-subtracted by subtracting either the first ramp in TRPV6-transfected cells or by averaging the first two to four ramps after establishing the whole-cell mode in untransfected cells (depending on how fast ICRAC developed) and then subtracting the mean from all subsequent traces. This type of analysis is well established for ICRAC measurements in RBL cells and was also used for TRPV6-expressing cells to allow a better comparison of the data in Figs. 2 and 3. No background current subtraction was performed for the data from TRPV6-expressing cells shown in Figs.1 A, 4 A, and Figure 5, Figure 6, Figure 7, Figure 8. The peak current amplitude was measured after leak subtraction at −80 mV for the data presented in the dose-response curve in Fig. 4 B. However, no reliable values could be determined for the lowest EGTA concentration tested (0.1 mm) and for the experiments with 100 nm[Ca2+]i and 1.05 mm[Mg2+]i (clamped by either 10 mm EGTA, 3.64 mm CaCl2, and 3.08 mm Mg2+ or 60 mm EGTA, 21.84 mm CaCl2, and 3.46 mmMg2+ in the pipette solution). No current activated under these conditions and amplitudes was measured 1 min after establishing the whole-cell configuration, because TRPV6 channels fully activate usually within this time. The global [Ca2+]i and [Mg2+]i were calculated by custom software (0.58, 1.16, 3.49, 11.66, 23.31, 34.97, 69.91, and 348.59 pm [Ca2+]i; and 0.64, 0.79, 0.95, 1.02, 1.03, 1.04, 1.04, and 1.05 mm[Mg2+]i at 60, 30, 10, 3, 1.5, 1, 0.5, and 0.1 mm EGTA, respectively). Throughout, average data are given as means ± S.E. for n cells. Student's paired t test was used for comparison of means.Figure 3Effects of store depletion at low intracellular Ca2+ buffering in TRPV6-expressing HEK and RBL cells in comparison to untransfected RBL cells.Ca2+ current activation in TRPV6-transfected HEK and RBL cells and nontransfected RBL cells. Cells were dialyzed with the internal solution containing either EGTA (1.5 mm) and IP3 (30 μm) or EGTA (0.1 mm), IP3 (30 μm), and thapsigargin (2 μm). Otherwise identical conditions as described for Fig.2. Mean data are shown after background current subtraction (1.5 mm EGTA, 30 μm IP3:n = 7 for HEK-TRPV6, n = 9 for RBL-TRPV6, n = 7 for RBL-wt; 0.1 mm EGTA, 30 μm IP3 and 2 μmthapsigargin: n = 3 for HEK-TRPV6, n = 10 for RBL-TRPV6, n = 8 for RBL-wt).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Current-voltage relationship from TRPV6-expressing HEK and nontransfected RBL cells. Whole-cell patch-clamp experiments were performed on TRPV6-transfected HEK cells and RBL wild-type cells as described under "Experimental Procedures." Currents were measured after maximum activation under identical conditions. The cells were dialyzed with a cesium glutamate-based internal solution containing 10 mm EGTA. A modified Ringer's solution with 10 mm Ca2+ was used extracellularly. The holding potential was −10 mV. Ashows the I-V relationship (mean ± S.E.) measured from a 4-ms average at the start (4 ms after the beginning of the voltage step) and the end of the pulse (4 ms prior to the end of the voltage step) as indicated by the arrows (n = 5). Theinset in A illustrates the voltage protocol from −110 mV to 10 mV for 200-s pulse duration and with 1 s between steps. Typical current traces are shown after maximal activation. Thedashed line in the lower figure of theinset represents zero current. B illustrates the voltage protocol from −110 mV to 90 mV in 50 ms. Representative current traces for a TRPV6-expressing HEK cell (black) and a nontransfected RBL cell (gray) are shown. Both current traces were leak-subtracted as described under "Experimental Procedures" and scaled to their maximum amplitude. Thedashed line represents zero current.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4TRPV6 channel activity depends on the intracellular EGTA concentration. A, activation of TRPV6-mediated Ca2+ currents in transfected HEK cells at various EGTA concentrations in the intracellular solution. No leak subtraction was performed. B, dose-response curve for Ca2+ entry in TRPV6-expressing HEK cells in dependence to the [EGTA]i. Data were averaged from 3, 3, 5, 7, 8, 7, 6, and 10 experiments for 0.1, 0.5, 1, 1.5, 3, 10, 30, and 60 mm [EGTA]i, respectively. Control experiments were performed on nontransfected HEK cells (n = 7, 5, 4, and 7 for 0.1, 0.5, 1.5, and 10 mm [EGTA]i). Values in the dose-response curve were obtained after background current subtraction. For details to the analysis, see "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8TRPV6-mediated monovalent currents in the absence of extracellular divalent cations. A, time course of representative currents from a TRPV6-expressing HEK cell dialyzed with an intracellular solution containing 10 mm EGTA and 6 mm Mg-ATP (n = 4). The normal external solution was switched to a divalent-free saline (DVF), as indicated by the bar. B, I-V relations were obtained during the experiment shown in A at the time pointshighlighted. None of the current traces were subtracted for initial conductances.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Current-voltage relationship from TRPV6-expressing HEK and nontransfected RBL cells in the absence of extracellular divalent cations. A, TRPV6-transfected HEK cells (n = 4) were dialyzed with an intracellular solution containing 10 mm EGTA and 6 mm Mg-ATP. No divalent cations were present in the bath solution (see "Experimental Procedures"). B, I-V relationship of a representative TRPV6-expressing HEK cell measured with the ramp protocol shown. The typical current trace of a RBL wild-type cell was recorded with an intracellular solution containing 30 μmIP3, 10 mm EGTA, and 6 mm Mg-ATP. All monovalent currents were recorded after maximal activation of the inward currents. No background current subtraction was performed throughout. Conditions were otherwise not different from the ones used for the recordings shown in Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6TRPV6-mediated Ca2+ currents in the presence of high intracellular concentrations of either EGTA or BAPTA. Figure shows Ca2+ current development in TRPV6-expressing HEK cells following dialysis with 30 EGTA or BAPTA. Mean data with S.E. are plotted versus time. The time courses of currents were not corrected for initial background conductances (n = 21 for 30 mm BAPTA,n = 6 for 30 mm EGTA).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5TRPV6 channel activity in dependence to [Ca2+] i and [Mg2+] i. Ca2+ currents were recorded from TRPV6-expressing HEK cells using intracellular solutions at two different [Ca2+]i and [Mg2+]i each. The standard pipette solution supplemented with 60 mm EGTA had calculated [Ca2+]i of 580 fm and [Mg2+]i was 637 μm(n = 10). The addition of 730 μmMgCl2 (1.73 mm MgCl2 and 2 mm Mg-ATP in total) to this solution increased [Mg2+]i to 1.05 mm, whereas the [Ca2+]i was unaffected (n = 11). The same [Mg2+]i was adjusted in the third intracellular solution (1.08 mm MgCl2 and 2 mm Mg-ATP), and the [Ca2+]i was clamped at 100 nm with 10 mm EGTA and 3.6 mmCaCl2 (n = 5). Averaged current traces are shown without correction for the initial current.View Large Image Figure ViewerDownload Hi-res image Download (PPT) All chemicals were purchased from Sigma, except BAPTA (Molecular Probes). In TRPV6-transfected HEK cells, a large current was elicited by voltage ramps when [Ca2+]i was buffered by 10 mm EGTA in the patch pipette (Fig.1). The inwardly rectifying current reversed at positive potentials (≥30 mV, n = 5). At potentials more negative to −40 mV the current showed a fast inactivation (time constant of 7.6 ± 1.3 ms at −100 mV,n = 5). No such current was recorded in the absence of external Ca2+ from TRPV6-transfected HEK cells (data not shown, n = 5). Mock and nontransfected cells exhibited a small background current with a linear I-V relationship (data not shown, n = 5 each). The biophysical properties of heterologously expressed TRPV6 channels are similar to those of endogenous store-operated channels in RBL cells (Fig. 1 B). Both currents show a strong inward rectification with a positive reversal potential, indicating a high Ca2+selectivity. It has been argued for the TRPV6 protein from rat that this channel is only store-operated when its expression level stands in a special relation to the native signal transduction machinery of CHO cells (12Yue L. Peng J.B. Hediger M.A. Clapham D.E. Nature. 2001; 410: 705-709Crossref PubMed Scopus (320) Google Scholar). This idea was tested by expressing the human clone in RBL cells, which show a much more pronounced ICRAC than CHO and HEK cells. Subsequently, RBL cells might offer a more native environment for store-operated channels than HEK cells. Whether this affects the gating of TRPV6 channels was tested in the following experiments. ICRAC is gated by the filling state of IP3-sensitive Ca2+ stores. It can be activated by store depletion obtained by intracellular perfusion of IP3, the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin, and/or EGTA. Using IP3 (30 μm) and EGTA (10 mm) in the internal solution resulted in rapid activation of ICRAC(Fig. 2). TRPV6-expressing HEK cells showed a rapid current activation followed by a slow inactivation until a steady-state level was reached (Fig. 2). This was also the case for TRPV6-expressing RBL cells; immediately after obtaining the whole-cell configuration, the Ca2+ current developed, reached a maximum, and subsequently decayed to a lower level (Fig. 2). Very similar results were obtained with thapsigargin (2 μm) instead of IP3 or both substances (TableI); ICRAC activated rapidly in RBL cells, no prominent current was detected in HEK cells, and a large inward current was measured in TRPV6-expressing HEK and RBL cells such as shown in Figs. 1 and 2. No significant differences between both cell types were detected with respect to the peak Ca2+current, activation, and inactivation kinetics.Table IPeak current amplitudes in TRPV6-expressing HEK and RBL cells under various intracellular conditionsHEK-TRPV6RBL-TRPV6EGTA + thapsigargin + IP3−17.0 ± 2.9 (6)−25.6 ± 4.6 (3)EGTA + thapsigargin−23.8 ± 4.2 (3)−25.2 ± 3.9 (3)EGTA + IP3−22.2 ± 5.7 (4)−26.3 ± 4.2 (5)EGTA−18.2 ± 2.7 (7)−19.1 ± 1.2 (3)Ca2+ currents were recorded from TRPV6-transfected HEK and RBL cells. The patch pipette was filled with the standard cesium glutamate-based solution supplemented with EGTA (10 mm), thapsigargin (2 μm), and IP3 (30 μm) as indicated. The bath solution contained 10 mM Ca2+. Cells were clamped at −10 mV, and maximal inward currents were measured at −80 mV during repetitive voltage ramps after background current subtraction as described under "Experimental Procedures." Mean data ± S.E. (n) are given. Open table in a new tab Ca2+ currents were recorded from TRPV6-transfected HEK and RBL cells. The patch pipette was filled with the standard cesium glutamate-based solution supplemented with EGTA (10 mm), thapsigargin (2 μm), and IP3 (30 μm) as indicated. The bath solution contained 10 mM Ca2+. Cells were clamped at −10 mV, and maximal inward currents were measured at −80 mV during repetitive voltage ramps after background current subtraction as described under "Experimental Procedures." Mean data ± S.E. (n) are given. The activation of ICRAC by high concentrations of the Ca2+ buffer EGTA (10 mm) in the intracellular solution is much slower than in the recordings with IP3and/or thapsigargin (Fig. 2). Strikingly, the activation kinetic of TRPV6-mediated Ca2+ currents was not changed in both expression systems tested. Similar results were obtained with BAPTA instead of EGTA (data not shown). Taken together, these data suggest that the presence of store-depleting agents such as thapsigargin and IP3 does not influence TRPV6 channel activation. It is possible to record ICRAC under conditions of moderate cytoplasmic Ca2+ buffering (20Zweifach A. Lewis R.S. J. Biol. Chem. 1995; 270: 14445-14451Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). With low concentrations of EGTA (1.5 mm) and the addition of IP3 (30 μm) in the pipette solution, peak current amplitude in RBL wild-type cells was similar to the recordings prese
Referência(s)