Lanthanides Potentiate TRPC5 Currents by an Action at Extracellular Sites Close to the Pore Mouth
2003; Elsevier BV; Volume: 278; Issue: 6 Linguagem: Inglês
10.1074/jbc.m211484200
ISSN1083-351X
AutoresSilke Jung, Anja Mühle, Michael Schaefer, Rainer Strotmann, Günter Schultz, Tim Plant,
Tópico(s)Respiratory and Cough-Related Research
ResumoMammalian members of the classical transient receptor potential channel (TRPC) subfamily (TRPC1–7) are Ca2+-permeable cation channels involved in receptor-mediated increases in intracellular Ca2+. Unlike most other TRP-related channels, which are inhibited by La3+ and Gd3+, currents through TRPC4 and TRPC5 are potentiated by La3+. Because these differential effects of lanthanides on TRPC subtypes may be useful for clarifying the role of different TRPCs in native tissues, we characterized the potentiating effect in detail and localized the molecular determinants of potentiation by mutagenesis. Whole cell currents through TRPC5 were reversibly potentiated by micromolar concentrations of La3+or Gd3+, whereas millimolar concentrations were inhibitory. By comparison, TRPC6 was blocked to a similar extent by La3+ or Gd3+ at micromolar concentrations and showed no potentiation. Dual effects of lanthanides on TRPC5 were also observed in outside-out patches. Even at micromolar concentrations, the single channel conductance was reduced by La3+, but reduction in conductance was accompanied by a dramatic increase in channel open probability, leading to larger integral currents. Neutralization of the negatively charged amino acids Glu543and Glu595/Glu598, situated close to the extracellular mouth of the channel pore, resulted in a loss of potentiation, and, for Glu595/Glu598 in a modification of channel inhibition. We conclude that in the micromolar range, the lanthanide ions La3+ and Gd3+ have opposite effects on whole cell currents through TRPC5 and TRPC6 channels. The potentiation of TRPC4 and TRPC5 by micromolar La3+ at extracellular sites close to the pore mouth is a promising tool for identifying the involvement of these isoforms in receptor-operated cation conductances of native cells. Mammalian members of the classical transient receptor potential channel (TRPC) subfamily (TRPC1–7) are Ca2+-permeable cation channels involved in receptor-mediated increases in intracellular Ca2+. Unlike most other TRP-related channels, which are inhibited by La3+ and Gd3+, currents through TRPC4 and TRPC5 are potentiated by La3+. Because these differential effects of lanthanides on TRPC subtypes may be useful for clarifying the role of different TRPCs in native tissues, we characterized the potentiating effect in detail and localized the molecular determinants of potentiation by mutagenesis. Whole cell currents through TRPC5 were reversibly potentiated by micromolar concentrations of La3+or Gd3+, whereas millimolar concentrations were inhibitory. By comparison, TRPC6 was blocked to a similar extent by La3+ or Gd3+ at micromolar concentrations and showed no potentiation. Dual effects of lanthanides on TRPC5 were also observed in outside-out patches. Even at micromolar concentrations, the single channel conductance was reduced by La3+, but reduction in conductance was accompanied by a dramatic increase in channel open probability, leading to larger integral currents. Neutralization of the negatively charged amino acids Glu543and Glu595/Glu598, situated close to the extracellular mouth of the channel pore, resulted in a loss of potentiation, and, for Glu595/Glu598 in a modification of channel inhibition. We conclude that in the micromolar range, the lanthanide ions La3+ and Gd3+ have opposite effects on whole cell currents through TRPC5 and TRPC6 channels. The potentiation of TRPC4 and TRPC5 by micromolar La3+ at extracellular sites close to the pore mouth is a promising tool for identifying the involvement of these isoforms in receptor-operated cation conductances of native cells. classical transient receptor potential channel 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid diacylglycerol enhanced green fluorescent protein human embryonic kidney N-methyl-d-glucamine the product of the number (N) of channels in the patch and the open probability 1-oleoyl-2-acetyl-sn-glycerol wild type yellow fluorescent protein Mammalian isoforms of the classical transient receptor potential channel (TRPC)1 subfamily, TRPC1–7, are likely candidates for cation channels mediating phospholipase C-dependent, receptor-operated Ca2+ influx (for reviews, see Refs. 1Harteneck C. Plant T.D. Schultz G. Trends Neurosci. 2000; 23: 159-166Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar, 2Montell, C. (2001) Science's STKEhttp://www.stke.org/cgi/content/full/OC_sigtrans;2001/90/re1Google Scholar, 3Montell C. Birnbaumer L. Flockerzi V. Cell. 2002; 108: 595-598Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar, 4Clapham D.E. Runnels L.W. Strübing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (947) Google Scholar, 5Zitt C. Halaszovich C.R. Lückhoff A. Prog. Neurobiol. 2002; 66: 243-264Crossref PubMed Scopus (122) Google Scholar). The properties and activation mechanisms of TRPCs have been studied extensively in heterologous expression systems. By contrast, relatively little information is available on their role in native cells. Two recent studies (6Inoue R. Okada T. Onoue H. Hara Y. Shimizu S. Naitoh S. Ito Y. Mori Y. Circ. Res. 2001; 88: 325-332Crossref PubMed Scopus (546) Google Scholar, 7Jung S. Strotmann R. Schultz G. Plant T.D. Am. J. Physiol. 2002; 282: C347-C359Crossref PubMed Scopus (222) Google Scholar) report that endogenous receptor-stimulated cation currents in vascular smooth muscle cells show properties identical to those described for heterologously expressed TRPC6. Therefore, precise knowledge of the properties of heterologously expressed TRPC channels may be essential to evaluate the involvement of TRPC proteins in receptor-operated cation conductances in native cells.Structurally, TRP channels, like many other cation channels, have been proposed to have six transmembrane segments (S1–S6), intracellular N and C termini, and a pore-forming reentrant loop between S5 and S6. Based on amino acid sequence similarity, the mammalian members of the TRPC subfamily can be subdivided into four groups (4Clapham D.E. Runnels L.W. Strübing C. Nat. Rev. Neurosci. 2001; 2: 387-396Crossref PubMed Scopus (947) Google Scholar,8Montell 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: 229-231Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar): TRPC1 (group 1), TRPC2 (group 2), TRPC3/6/7 (group 3), and TRPC4/5 (group 4). This subdivision is also supported by functional data. One of the major functional criteria is the mechanism of channel activation. From studies in heterologous expression systems, it is undisputed that receptor-mediated stimulation of phospholipase C is a key event in the activation of all TRPC isoforms. Evidence indicates that currents mediated by TRPC3, TRPC6, or TRPC7 can be activated by diacylglycerol (DAG) independently of protein kinase C (9Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Crossref PubMed Scopus (1237) Google Scholar, 10Ma H.T. Patterson R.L. van Rossum D.B. Birnbaumer L. Mikoshiba K. Gill D.L. Science. 2000; 287: 1647-1651Crossref PubMed Scopus (531) Google Scholar, 11Venkatachalam K., Ma, H.T. Ford D.L. Gill D.L. J. Biol. Chem. 2001; 276: 33980-33985Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 12Zhang L. Saffen D. J. Biol. Chem. 2001; 276: 13331-13339Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), although there is some controversy regarding the physiological significance of this stimulation (13Kiselyov K. Muallem S. Trends Neurosci. 1999; 22: 334-337Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). By contrast, TRPC4 and TRPC5 are not activated by DAG, and some evidence indicates that unidentified components of the phospholipase C pathway other than DAG or store depletion activate the channels (14Okada T. Shimizu S. Wakamori M. Maeda A. Kurosaki T. Takada N. Imoto K. Mori Y. J. Biol. Chem. 1998; 273: 10279-10287Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 15Schaefer M. Plant T.D. Obukhov A.G. Hofmann T. Gudermann T. Schultz G. J. Biol. Chem. 2000; 275: 17517-17526Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 16Strübing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar). Other evidence supports a store-dependent activation mechanism (17Philipp S. Hambrecht J. Braslavski L. Schroth G. Freichel M. Murakami M. Cavalié A. Flockerzi V. EMBO J. 1998; 17: 4274-4282Crossref PubMed Scopus (271) Google Scholar, 18Tomita Y. Kaneko S. Funayama M. Kondo H. Satoh M. Akaike A. Neurosci. Lett. 1998; 248: 195-198Crossref PubMed Scopus (44) Google Scholar, 19Warnat J. Philipp S. Zimmer S. Flockerzi V. Cavalié A. J. Physiol. 1999; 518: 631-638Crossref PubMed Scopus (87) Google Scholar, 20Kinoshita M. Akaike A. Satoh M. Kaneko S. Cell Calcium. 2000; 28: 151-159Crossref PubMed Scopus (19) Google Scholar). TRPC1 has been reported to be a store-dependent channel in some studies (21Zitt C. Zobel A. Obukhov A.G. Harteneck C. Kalkbrenner F. Lückhoff A. Schultz G. Neuron. 1996; 16: 1189-1196Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 22Liu X. Wang W. Singh B.B. Lockwich T. Jadlowiec J. O'Connell B. Wellner R. Zhu M.X. Ambudkar I.S. J. Biol. Chem. 2000; 275: 3403-3411Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 23Delmas P. Wanaverbecq N. Abogadie F.C. Mistry M. Brown D.A. Neuron. 2002; 34: 209-220Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), but doubts have been raised as to whether TRPC1 expression results in the formation of functional plasma membrane channels in mammalian cells (16Strübing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 24Sinkins W.G. Estacion M. Schilling W.P. Biochem. J. 1998; 331: 331-339Crossref PubMed Scopus (93) Google Scholar, 25Hofmann T. Schaefer M. Schultz G. Gudermann T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7461-7466Crossref PubMed Scopus (625) Google Scholar). The few data available support a store-operated activation mechanism for TRPC2 (26Vannier B. Peyton M. Boulay G. Brown D. Qin N. Jiang M. Zhu X. Birnbaumer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2060-2064Crossref PubMed Scopus (234) Google Scholar, 27Jungnickel M.K. Marrero H. Birnbaumer L. Lemos J.R. Florman H.M. Nat. Cell Biol. 2001; 3: 499-502Crossref PubMed Scopus (277) Google Scholar), but, here again, other evidence suggests that TRPC2 does not form functional channels in all tissues (28Hofmann T. Schaefer M. Schultz G. Gudermann T. Biochem. J. 2000; 351: 115-122Crossref PubMed Scopus (75) Google Scholar). Data from our group, confirmed in several independent laboratories, indicate that several biophysical features are also characteristic of certain groups of TRPCs. Thus, TRPC3–7 have a characteristic doubly rectifying, or S-shaped current-voltage relation (9Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Crossref PubMed Scopus (1237) Google Scholar, 14Okada T. Shimizu S. Wakamori M. Maeda A. Kurosaki T. Takada N. Imoto K. Mori Y. J. Biol. Chem. 1998; 273: 10279-10287Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 15Schaefer M. Plant T.D. Obukhov A.G. Hofmann T. Gudermann T. Schultz G. J. Biol. Chem. 2000; 275: 17517-17526Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 16Strübing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 29Okada T. Inoue R. Yamazaki K. Maeda A. Kurosaki T. Yamakuni T. Tanaka I. Shimizu S. Ikenaka K. Imoto K. Mori Y. J. Biol. Chem. 1999; 274: 27359-27370Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 30Kiselyov K.I. Shin D.M. Wang Y. Pessah I.N. Allen P.D. Muallem S. Mol. Cell. 2000; 6: 421-431Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 31Schaefer M. Plant T.D. Stresow N. Albrecht N. Schultz G. J. Biol. Chem. 2002; 277: 3752-3759Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Furthermore, at the single channel level, even though the amplitudes of single channel events are similar, the openings of TRPC3 and TRPC6 are very brief (9Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Crossref PubMed Scopus (1237) Google Scholar, 32Zitt C. Obukhov A.G. Strübing C. Zobel A. Kalkbrenner F. Lückhoff A. Schultz G. J. Cell Biol. 1997; 138: 1333-1341Crossref PubMed Scopus (221) Google Scholar, 33Hurst R.S. Zhu X. Boulay G. Birnbaumer L. Stefani E. FEBS Lett. 1998; 422: 333-338Crossref PubMed Scopus (95) Google Scholar, 34Kiselyov K., Xu, X. Mozhayeva G. Kuo T. Pessah I. Mignery G. Zhu X. Birnbaumer L. Muallem S. Nature. 1998; 396: 478-482Crossref PubMed Scopus (558) Google Scholar, 35Kiselyov K. Mignery G.A. Zhu M.X. Muallem S. Mol. Cell. 1999; 4: 423-429Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) compared with those of TRPC4 and TRPC5 (15Schaefer M. Plant T.D. Obukhov A.G. Hofmann T. Gudermann T. Schultz G. J. Biol. Chem. 2000; 275: 17517-17526Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 16Strübing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 31Schaefer M. Plant T.D. Stresow N. Albrecht N. Schultz G. J. Biol. Chem. 2002; 277: 3752-3759Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 36Yamada H. Wakamori M. Hara Y. Takahashi Y. Konishi K. Imoto K. Mori Y. Neurosci. Lett. 2000; 285: 111-114Crossref PubMed Scopus (35) Google Scholar).In the absence of more specific pharmacological tools, the lanthanides lanthanum (La3+) and gadolinium (Gd3+) are commonly used blockers of nonselective cation channels and other Ca2+-permeable channels. Interestingly, recent studies have reported that 100 μm La3+ has potentiating effects on mouse, rat, and human TRPC4 and mouse TRPC5 (15Schaefer M. Plant T.D. Obukhov A.G. Hofmann T. Gudermann T. Schultz G. J. Biol. Chem. 2000; 275: 17517-17526Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 16Strübing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 31Schaefer M. Plant T.D. Stresow N. Albrecht N. Schultz G. J. Biol. Chem. 2002; 277: 3752-3759Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). However, the actions of La3+ on TRPC4 and TRPC5 have not been characterized further. By contrast, other TRPCs are inhibited by micromolar concentrations of La3+ or Gd3+ (6Inoue R. Okada T. Onoue H. Hara Y. Shimizu S. Naitoh S. Ito Y. Mori Y. Circ. Res. 2001; 88: 325-332Crossref PubMed Scopus (546) Google Scholar,29Okada T. Inoue R. Yamazaki K. Maeda A. Kurosaki T. Yamakuni T. Tanaka I. Shimizu S. Ikenaka K. Imoto K. Mori Y. J. Biol. Chem. 1999; 274: 27359-27370Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 37Zhu X. Jiang M. Birnbaumer L. J. Biol. Chem. 1998; 273: 133-142Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 38Kamouchi M. Philipp S. Flockerzi V. Wissenbach U. Mamin A. Raeymaekers L. Eggermont J. Droogmans G. Nilius B. J. Physiol. 1999; 518: 345-358Crossref PubMed Scopus (162) Google Scholar, 39Halaszovich C.R. Zitt C. Jüngling E. Lückhoff A. J. Biol. Chem. 2000; 275: 37423-37428Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 40Riccio A. Mattei C. Kelsell R.E. Medhurst A.D. Calver A.R. Randall A.D. Davis J.B. Benham C.D. Pangalos M.N. J. Biol. Chem. 2002; 277: 12302-12309Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar).Because the different effects of lanthanides are potentially an important distinguishing feature of the group 4 TRPC channels, we characterized the effect of these ions on TRPC5 in detail and compared them with those on TRPC6, a member of group 3. For this study we chose the rat TRPC6B slice variant (12Zhang L. Saffen D. J. Biol. Chem. 2001; 276: 13331-13339Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), which lacks 54 amino acids at the distal N terminus compared with rat TRPC6A, and has not previously been characterized electrophysiologically. Unlike TRPC6A, TRPC6B has been reported to be activated by agonist application but not by 1-oleoyl-2-acetyl-sn-glycerol (OAG) (12Zhang L. Saffen D. J. Biol. Chem. 2001; 276: 13331-13339Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In whole cell patch clamp recordings, we found that TRPC5 was bimodally modulated by lanthanides, with potentiation at micromolar concentrations being succeeded by inhibition at millimolar concentrations. In contrast, TRPC6 was inhibited by micromolar concentrations and showed no potentiation. At the single channel level, the effects of La3+ on TRPC5 are complex, affecting the single channel conductance, the mean open time, and the frequency of channel openings. By site-specific neutralization of extracellular negatively charged amino acids, we have identified two sites, close to the pore mouth, that are involved in potentiation of TRPC5 by La3+.DISCUSSIONIn the present study, we show that currents mediated by TRPC5 and TRPC6 are affected differently by the lanthanides La3+ and Gd3+. Although whole cell currents through TRPC6 were inhibited concentration-dependently by lanthanides, those through TRPC5 were potentiated by low concentrations but inhibited by high concentrations. The dual effect of La3+ on TRPC5 was also observed at the single channel level and involved a combination of an inhibitory effect on channel amplitude and an increase in channel open probability. By an analysis of point mutants, we identified two sites, close to the extracellular mouth of the pore, which are involved in La3+- and Ca2+-induced channel potentiation.Inhibitory effects of different di- and trivalent cations, including La3+ and Gd3+, have been described for most Ca2+-permeable channels. Accordingly, current block by bath application of lanthanide ions has been reported for several members of the TRPC subfamily of TRP channels, e.g. for human and mouse TRPC3 (29Okada T. Inoue R. Yamazaki K. Maeda A. Kurosaki T. Yamakuni T. Tanaka I. Shimizu S. Ikenaka K. Imoto K. Mori Y. J. Biol. Chem. 1999; 274: 27359-27370Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 37Zhu X. Jiang M. Birnbaumer L. J. Biol. Chem. 1998; 273: 133-142Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 38Kamouchi M. Philipp S. Flockerzi V. Wissenbach U. Mamin A. Raeymaekers L. Eggermont J. Droogmans G. Nilius B. J. Physiol. 1999; 518: 345-358Crossref PubMed Scopus (162) Google Scholar, 39Halaszovich C.R. Zitt C. Jüngling E. Lückhoff A. J. Biol. Chem. 2000; 275: 37423-37428Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), for mouse TRPC6 (6Inoue R. Okada T. Onoue H. Hara Y. Shimizu S. Naitoh S. Ito Y. Mori Y. Circ. Res. 2001; 88: 325-332Crossref PubMed Scopus (546) Google Scholar, 45Boulay G. Zhu X. Peyton M. Jiang M. Hurst R. Stefani E. Birnbaumer L. J. Biol. Chem. 1997; 272: 29672-29680Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar), and for human TRPC7 (40Riccio A. Mattei C. Kelsell R.E. Medhurst A.D. Calver A.R. Randall A.D. Davis J.B. Benham C.D. Pangalos M.N. J. Biol. Chem. 2002; 277: 12302-12309Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). There is considerable variability in the IC50 values obtained, with higher values in Ca2+ imaging experiments than in electrophysiological recordings. The IC50 value obtained for rat TRPC6 in the present study was in good agreement with the values obtained in whole cell patch clamp experiments for mouse TRPC6 (6Inoue R. Okada T. Onoue H. Hara Y. Shimizu S. Naitoh S. Ito Y. Mori Y. Circ. Res. 2001; 88: 325-332Crossref PubMed Scopus (546) Google Scholar) and human TRPC3 (39Halaszovich C.R. Zitt C. Jüngling E. Lückhoff A. J. Biol. Chem. 2000; 275: 37423-37428Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar).The pronounced increase in agonist-induced currents in TRPC5-expressing cells with micromolar La3+ in the present study are in agreement with previous studies on this channel (15Schaefer M. Plant T.D. Obukhov A.G. Hofmann T. Gudermann T. Schultz G. J. Biol. Chem. 2000; 275: 17517-17526Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 16Strübing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar), and we have extended this observation to Gd3+, which is approximately equally effective. A further novel finding of the present study is that higher lanthanide concentrations (≥1 mm) were less effective in potentiating the current and even reversibly inhibited currents carried by TRPC5. Millimolar concentrations of Ca2+ also potentiated agonist-induced currents and prevented the effects of micromolar La3+, suggesting that physiological divalent cations may also bind at the same site.Evidence for two different actions of lanthanides on the channel was supported by the effects of La3+ on single channel currents in outside-out patches. La3+ caused a concentration-dependent decrease in single channel current amplitude, while, at the same time, increasing the channel open probability (NPo). Both effects were already observed at a concentration of 1 μm. The concentration dependence of the increase in current in outside-out patches (NPo·i) closely paralleled the increase in whole cell current, although the maximum potentiation was, on average, about 3–4-fold higher in outside-out patches than in whole cell experiments. Because of our inability to resolve currents at millimolar concentrations of La3+, it is not clear from the single channel data why potentiation declines and inhibition occurs. A decrease in single channel current is at least partly responsible for the decrease in whole cell current.There are few reports of potentiating effects of La3+ on ion channel currents, and more importantly, to our knowledge, there are no reports that describe dual effects on ion channel activity. Potentiating actions of 100 μm La3+ have been observed in whole cell recordings for mouse, rat, and human TRPC4- and mouse TRPC5-mediated currents (15Schaefer M. Plant T.D. Obukhov A.G. Hofmann T. Gudermann T. Schultz G. J. Biol. Chem. 2000; 275: 17517-17526Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar, 16Strübing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 31Schaefer M. Plant T.D. Stresow N. Albrecht N. Schultz G. J. Biol. Chem. 2002; 277: 3752-3759Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) and for receptor-operated currents in cells coexpressing mouse TRPC1 and mouse TRPC5 (16Strübing C. Krapivinsky G. Krapivinsky L. Clapham D.E. Neuron. 2001; 29: 645-655Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar). In the latter, heteromultimers of TRPC1 and TRPC5 are thought to be formed, which, compared with homomeric TRPC5, have a drastically reduced single channel current (−0.5 pA at a holding potential of −60 mV). The single channel current amplitude was not affected by the inclusion of La3+ in the pipette solution. For native nonselective cation currents, there is one report of a potentiation of the native current (I cat) in rat ileal smooth muscle cells by La3+, with an apparent Kd of 190 μm (46Inoue R. Morita H. Yanagida H. Ito Y. J. Smooth Muscle Res. 1998; 34: 69-81Crossref PubMed Scopus (14) Google Scholar). From relaxation analysis, prolonged single channel mean open life times were suggested to be the main cause of the augmentative effect of La3+. Interestingly, in the mouse, TRPC4 is expressed in this tissue (47Walker R.L. Hume J.R. Horowitz B. Am. J. Physiol. 2001; 280: C1184-C1192Crossref PubMed Google Scholar). With regard to heterologously expressed TRPC4 and TRPC5, it should be noted that some studies reported an inhibition by micromolar lanthanide concentrations (14Okada T. Shimizu S. Wakamori M. Maeda A. Kurosaki T. Takada N. Imoto K. Mori Y. J. Biol. Chem. 1998; 273: 10279-10287Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar,48McKay R.R. Szymeczek-Seay C.L. Lievremont J.P. Bird G.S. Zitt C. Jüngling E. Lückhoff A. Putney J.W. Biochem. J. 2000; 351: 735-746Crossref PubMed Scopus (113) Google Scholar).The loss of the potentiating effects of La3+ and Ca2+ in mutants of two sites (Glu543 and Glu595/Glu598), which, according to models of TRP channel structure, are located opposite each other at the start and end of the pore-forming loop between S5 and S6, strongly supports an extracellular site of action. Importantly, identical amino acids are present in TRPC4 at the positions corresponding to Glu543and Glu595 in TRPC5, but acid amino acids are not present at corresponding positions in TRPC3, TRPC6, and TRPC7. Larger variations in structure prevent an identification of corresponding residues in TRPC1. The differences between the TRPC isoforms provide an explanation for the specificity of the potentiating effect for TRPC4 and TRPC5. Interestingly, these sites are analogous to those in TRPV1 (VR1) which are involved in proton-mediated channel potentiation (Glu600) and proton-mediated channel activation (Glu648) (49Jordt S.E. Tominaga M. Julius D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8134-8139Crossref PubMed Scopus (526) Google Scholar) and can modulate sensitivity to the activator capsaicin (49Jordt S.E. Tominaga M. Julius D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8134-8139Crossref PubMed Scopus (526) Google Scholar, 50Welch J.M. Simon S.A. Reinhart P.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13889-13894Crossref PubMed Scopus (168) Google Scholar). Indeed, at the latter site TRPC4, TRPC5, and TRPV1 have identical EFTE motifs. Because the distal steps leading to activation of this channel and the activation mechanism are not known, it is not clear how La3+ or Ca2+ binding to the extracellular sites results in current potentiation. By analogy to TRPV1, where neutralization of Glu600 and Glu648 leads to potentiation of the capsaicin sensitivity (49Jordt S.E. Tominaga M. Julius D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8134-8139Crossref PubMed Scopus (526) Google Scholar, 50Welch J.M. Simon S.A. Reinhart P.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13889-13894Crossref PubMed Scopus (168) Google Scholar), it is tempting to speculate that La3+ or Ca2+, by neutralizing the negative charges, potentiate the response of TRPC5 to its unknown activator.The TRPC5 mutation E595Q/E598Q also affected inhibition by La3+, whereas E543Q did not. For the wild type channel, the reduction in single channel current by La3+ at all potentials without an increase in open channel noise is indicative of a fast block at a site outside the membrane electrical field. Similarly, inhibition of whole cell currents in the mutant E543Q, which lacked potentiation, was potential-independent. In contrast, the mutant E595Q/E598Q showed a slower flickery block at the single channel level and a clear potential dependence of whole cell current inhibition, with inward currents being more strongly reduced than outward currents. The loss of the fast block by mutation at the extracellular site E595Q/E598Q and the potential dependence of the block remaining after mutation indicate that in both cases La3+ blocks the channel from the outside. The effect of this mutation on inhibition by La3+, the increase in single channel current, and the increase in P Ca/P Nasuggest that this site lies close to, or in, the permeation pathway. Considering the change in channel inhibition by La3+, it is possible that the increase in single channel current in E595Q/E598Q results from a reduction in block by a physiological cation. By analogy to other channels with similar structure, the glutamates will form a negatively charged ring around the extracellular pore mouth, with, in tetramers, at least 12 negatively charged residues. These amino acids may act as “gatekeepers” controlling cation entry into the pore.Further evidence that both potentiation of TRPC5 and inhibition of TRPC6 by lanthanides results from an extracellular action of La3+ is provided by the presence of the effects in experiments with intracellular EGTA buffers and their persistence in the presence of higher concentrations of BAPTA. Both buffers have a very high affinity for lanthanides. Recently, Halaszovich et al. (39Halaszovich C.R. Zitt C. Jüngling E. Lückhoff A. J. Biol. Chem. 2000; 275: 37423-37428Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) suggested that La3+ and Gd3+block human TRPC3 channels from the cytosolic side of the membrane and that different apparent IC50 values might simply reflect different uptake rates for lanthanide ions in different cell types (see below). Our data support an extracellular site of action on TRPC6 and on TRPC5, although we cannot exclude additional intracellular effects.Because of the variability in results from different laboratories, the applicability of results from heterologous overexpression studies on TRPC channels to native channels has recently been questioned (e.g. 5). However, for TRPC6, at least, properties nearly identical to those observed after overexpression are seen for native channels in vascular smooth muscle cells (6Inoue R. Okada T. Onoue H. Hara Y. Shimizu S. Naitoh S. Ito Y. Mori Y. Circ. Res. 20
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