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• W2072886932 abstract "L-type dihydropyridine-sensitive voltage dependent Ca2+ channels (L-VDCCs; α1C) are crucial in cardiovascular physiology. Currents via L-VDCCs are enhanced by hormones and transmitters operating via Gq, such as angiotensin II (AngII) and acetylcholine (ACh). It has been proposed that these modulations are mediated by protein kinase C (PKC). However, reports on effects of PKC activators on L-type channels are contradictory; inhibitory and/or enhancing effects have been observed. Attempts to reproduce the enhancing effect of AngII in heterologous expression systems failed. We previously found that PKC modulation of the channel depends on α1C isoform used; only a long N-terminal (NT) isoform was up-regulated. Here we report the reconstitution of the AngII- and ACh-induced enhancement of the long-NT isoform of L-VDCC expressed in Xenopus oocytes. The current initially increased over several minutes but later declined to below baseline levels. Using different NT deletion mutants and human short- and long-NT isoforms of the channel, we found the initial segment of the NT to be crucial for the enhancing, but not for the inhibitory, effect. Using blockers of PKC and of phospholipase C (PLC) and a mutated AngII receptor lacking Gq coupling, we demonstrate that the signaling pathway of the enhancing effect includes the activation of Gq, PLC, and PKC. The inhibitory modulation, present in both α1C isoforms, was Gq- and PLC-independent and Ca2+-dependent, but not Ca2+-mediated, as only basal levels of Ca2+ were essential. Reconstitution of AngII and ACh effects in Xenopus oocytes will advance the study of molecular mechanisms of these physiologically important modulations. L-type dihydropyridine-sensitive voltage dependent Ca2+ channels (L-VDCCs; α1C) are crucial in cardiovascular physiology. Currents via L-VDCCs are enhanced by hormones and transmitters operating via Gq, such as angiotensin II (AngII) and acetylcholine (ACh). It has been proposed that these modulations are mediated by protein kinase C (PKC). However, reports on effects of PKC activators on L-type channels are contradictory; inhibitory and/or enhancing effects have been observed. Attempts to reproduce the enhancing effect of AngII in heterologous expression systems failed. We previously found that PKC modulation of the channel depends on α1C isoform used; only a long N-terminal (NT) isoform was up-regulated. Here we report the reconstitution of the AngII- and ACh-induced enhancement of the long-NT isoform of L-VDCC expressed in Xenopus oocytes. The current initially increased over several minutes but later declined to below baseline levels. Using different NT deletion mutants and human short- and long-NT isoforms of the channel, we found the initial segment of the NT to be crucial for the enhancing, but not for the inhibitory, effect. Using blockers of PKC and of phospholipase C (PLC) and a mutated AngII receptor lacking Gq coupling, we demonstrate that the signaling pathway of the enhancing effect includes the activation of Gq, PLC, and PKC. The inhibitory modulation, present in both α1C isoforms, was Gq- and PLC-independent and Ca2+-dependent, but not Ca2+-mediated, as only basal levels of Ca2+ were essential. Reconstitution of AngII and ACh effects in Xenopus oocytes will advance the study of molecular mechanisms of these physiologically important modulations. The cardiac voltage-dependent, dihydropyridine-sensitive L-type calcium channel (L-VDCC) 1The abbreviations used are: L-VDCC, L-type voltage dependent Ca2+ channels; aa, amino acid; ACh, acetylcholine; AngII, angiotensin II; AT1R, angiotensin II receptor type 1; Bis, bis-indolylmaleimide; m1R and m3R, muscarinic receptors 1 and 3; NT, N-terminus; PKC, protein kinase C; PLC, phospholipase C; WT, wild-type; PMA, 4β-phorbol-12-myristate 13-acetate; ANOVA, analysis of variance; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. is the main calcium channel in the heart, where it contributes to the plateau of the action potential and thereby promotes cardiac cell contraction (1Reuter H. Nature. 1983; 301: 569-574Crossref PubMed Scopus (863) Google Scholar). In smooth muscle cells, these channels regulate tonus and contraction (2Hughes A.D. J. Vasc. Res. 1995; 32: 353-370Crossref PubMed Scopus (86) Google Scholar, 3Xiong Z. Sperelakis N. J. Mol. Cell. Cardiol. 1995; 27: 75-91Abstract Full Text PDF PubMed Scopus (124) Google Scholar). Different hormones and transmitters, such as angiotensin II (AngII), bradykinin, acetylcholine (ACh), and norepinephrine, modulate the function of L-VDCC via G-proteins and protein kinases, profoundly affecting the function of the corresponding tissues (4Trautwein W. Hescheler J. Annu. Rev. Physiol. 1990; 52: 257-274Crossref PubMed Scopus (318) Google Scholar). AngII and ACh activate Gq-coupled receptors and are involved in cardiovascular function, regulation of blood pressure, and renal function (5Berk B.C. Corson M.A. Circ. Res. 1997; 80: 607-616Crossref PubMed Scopus (284) Google Scholar, 6Beech D.J. Pharmacol. Ther. 1997; 73: 91-119Crossref PubMed Scopus (79) Google Scholar). In the heart, ACh inhibits L-VDCC via m2 muscarinic receptors and the subsequent activation of the Gi signaling cascade and inhibition of adenylyl cyclase (1Reuter H. Nature. 1983; 301: 569-574Crossref PubMed Scopus (863) Google Scholar). However, in the smooth muscle, both AngII and ACh are potent vasoconstrictors that both induce Ca2+ release from intracellular stores and elevate intracellular Ca2+ concentration (7Jackson E.K. Garrison J.C. Hardman J.G. Limbird L.E. Molinoff P.B. Ruddon R.W. Gilman A.G. Goodman & Gilman's The Pharmacological Basis of Therapeutics. McGraw-Hill, New York1996: 733-758Google Scholar, 8Eglen R.M. Reddy H. Watson N. Challiss R.A. Trends Pharmacol. Sci. 1994; 15: 114-119Abstract Full Text PDF PubMed Scopus (237) Google Scholar). In heart and smooth muscle, AngII enhances Ca2+ channel currents (9Takenaka T. Forster H. Epstein M. Circ. Res. 1993; 73: 743-750Crossref PubMed Scopus (45) Google Scholar, 10Scholz H. Kurtz A. Am. J. Physiol. 1990; 259: C421-C426Crossref PubMed Google Scholar, 11Purdy R.E. Weber M.A. Circ. Res. 1988; 63: 748-757Crossref PubMed Scopus (63) Google Scholar, 12Dosemeci A. Dhallan R.S. Cohen N.M. Lederer W.J. Rogers T.B. Circ. Res. 1988; 62: 347-357Crossref PubMed Scopus (215) Google Scholar, 13Macrez-Lepretre N. Morel J.L. Mironneau J. J. Pharmacol. Exp. Ther. 1996; 278: 468-475PubMed Google Scholar). ACh has also been reported to increase L-type Ca2+ channel currents in smooth muscle, mainly via m3 muscarinic receptors, m3R (14Benham C.D. Bolton T.B. Lang R.J. Nature. 1985; 316: 345-347Crossref PubMed Scopus (195) Google Scholar, 15Shi X.Z. Sarna S.K. Am. J. Physiol. 2000; 278: G234-G242PubMed Google Scholar, 16Liu X. Rusch N.J. Striessnig J. Sarna S.K. Gastroenterology. 2001; 120: 480-489Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Despite the clinical and physiological importance of the regulation of L-type Ca2+ channels by AngII and ACh, the molecular mechanisms remain poorly understood. The mechanism of AngII effect on L-type Ca2+ channels has been extensively studied but remains unclear and even controversial. Protein kinase C (PKC) is the most obvious and important mediator of AngII and ACh/m3R action. PKC is activated in native cells following AngII and ACh binding to Gq-coupled receptors (AngII receptor type 1, AT1R, and muscarinic receptors m3R and m1R, respectively). In mammals, PKC inhibitors block AngII-induced vasoconstriction (17Bauer J. Dau C. Cavarape A. Schaefer F. Ehmke H. Parekh N. Am. J. Physiol. 1999; 277: H1-H7PubMed Google Scholar, 18Nagahama T. Hayashi K. Ozawa Y. Takenaka T. Saruta T. Kidney Int. 2000; 57: 215-223Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 19Lambert C. Br. J. Pharmacol. 1995; 115: 795-800Crossref PubMed Scopus (19) Google Scholar, 20Fan Q.I. Vanderpool K. Marsh J.D. Biochim. Biophys. Acta. 2002; 1577: 401-411Crossref PubMed Scopus (12) Google Scholar). In cardiomyocytes and smooth muscle cells, the PKC activators phorbol esters and diacylglycerol mimic the effect of AngII, increasing force of contraction and Ca2+ influx (10Scholz H. Kurtz A. Am. J. Physiol. 1990; 259: C421-C426Crossref PubMed Google Scholar, 21Kato H. Hayashi T. Koshino Y. Kutsumi Y. Nakai T. Miyabo S. Biochem. Biophys. Res. Commun. 1992; 188: 934-941Crossref PubMed Scopus (32) Google Scholar, 22Savignac M. Badou A. Moreau M. Leclerc C. Guery J.C. Paulet P. Druet P. Ragab-Thomas J. Pelletier L. FASEB J. 2001; 15: 1577-1579Crossref PubMed Scopus (51) Google Scholar), as well as Ca2+ currents via the L-type channel (12Dosemeci A. Dhallan R.S. Cohen N.M. Lederer W.J. Rogers T.B. Circ. Res. 1988; 62: 347-357Crossref PubMed Scopus (215) Google Scholar, 23Fish R.D. Sperti G. Colucci W.S. Clapham D.E. Circ. Res. 1988; 62: 1049-1054Crossref PubMed Scopus (163) Google Scholar, 24Lacerda A.E. Rampe D. Brown A.M. Nature. 1988; 335: 249-251Crossref PubMed Scopus (211) Google Scholar, 25Tseng G.N. Boyden P.A. Am. J. Physiol. 1991; 261: H364-H379Crossref PubMed Google Scholar, 26Liu Q.Y. Karpinski E. Pang P.K. Biochem. Biophys. Res. Commun. 1993; 191: 796-801Crossref PubMed Scopus (27) Google Scholar, 27Schuhmann K. Groschner K. FEBS Lett. 1994; 341: 208-212Crossref PubMed Scopus (52) Google Scholar, 28Chik C.L. Li B. Ogiwara T. Ho A.K. Karpinski E. FASEB J. 1996; 10: 1310-1317Crossref PubMed Scopus (48) Google Scholar, 29Reeve H.L. Vaughan P.F. Peers C. Pfluegers Arch. Eur. J. Physiol. 1995; 429: 729-737Crossref PubMed Scopus (21) Google Scholar, 30Love J.A. Richards N.W. Owyang C. Dawson D.C. Am. J. Physiol. 1998; 274: G397-G405PubMed Google Scholar). This enhancement is sometimes followed by a later reduction in the current (24Lacerda A.E. Rampe D. Brown A.M. Nature. 1988; 335: 249-251Crossref PubMed Scopus (211) Google Scholar, 25Tseng G.N. Boyden P.A. Am. J. Physiol. 1991; 261: H364-H379Crossref PubMed Google Scholar, 31Boixel C. Tessier S. Pansard Y. Lang-Lazdunski L. Mercadier J.J. Hatem S.N. Am. J. Physiol. 2000; 278: H670-H676PubMed Google Scholar). In some cases, only an inhibition of the current in response to PKC activation has been reported (32Zhang Z.H. Johnson J.A. Chen L. El-Sherif N. Mochly-Rosen D. Boutjdir M. Circ. Res. 1997; 80: 720-729Crossref PubMed Scopus (84) Google Scholar). In addition to PKC, protein tyrosine kinases (33Watts S.W. Florian J.A. Monroe K.M. J. Pharmacol. Exp. Ther. 1998; 286: 1431-1438PubMed Google Scholar, 34Seki T. Yokoshiki H. Sunagawa M. Nakamura M. Sperelakis N. Pfluegers Arch. Eur. J. Physiol. 1999; 437: 317-323Crossref PubMed Scopus (58) Google Scholar) and the Gβγ dimer, via activation of phosphoinositol-3-kinase (35Quignard J.F. Mironneau J. Carricaburu V. Fournier B. Babich A. Nurnberg B. Mironneau C. Macrez N. J. Biol. Chem. 2001; 276: 32545-32551Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), have been implicated as being potentially involved in the mediation of AngII effects. A major obstacle in studying the molecular mechanisms of AngII and ACh modulations was that these modulations could not be reconstituted in heterologous expression systems. Bouron et al. (36Bouron A. Soldatov N.M. Reuter H. FEBS Lett. 1995; 377: 159-162Crossref PubMed Scopus (37) Google Scholar) studied the modulation of human L-VDCC expressed in Xenopus oocytes and reported that following the administration of PMA, the dihydropyridine-sensitive IBa was inhibited. Oz et al. (37Oz M. Melia M.T. Soldatov N.M. Abernethy D.R. Morad M. Mol. Pharmacol. 1998; 54: 1106-1112Crossref PubMed Scopus (46) Google Scholar) reported a decrease in Ca2+ current in oocytes expressing α1C and AT1R following application of AngII, due to a Ca2+-dependent mechanism. This effect was blocked by chelating Ca2+ or by depleting intracellular Ca2+ stores with thapsigargin. The failure to reproduce AngII-induced Ca2+ channel enhancement may be related to the use of certain isoforms of α1C that are not modulated by PKC. In the rat and rabbit, two N-terminal (NT) isoforms of α1C (Cav2.1) are known, which probably represent variable splicing products of the same gene (38Snutch T.P. Tomlinson W.J. Leonard J.P. Gilbert M.M. Neuron. 1991; 7: 45-57Abstract Full Text PDF PubMed Scopus (296) Google Scholar). These splice variants encode long- and short-NT α1C proteins, with variable initial segments of 46 and 16 amino acids (aa), respectively (38Snutch T.P. Tomlinson W.J. Leonard J.P. Gilbert M.M. Neuron. 1991; 7: 45-57Abstract Full Text PDF PubMed Scopus (296) Google Scholar, 39Mikami A. Imoto K. Tanabe T. Niidome T. Mori Y. Takeshima H. Narumiya S. Numa S. Nature. 1989; 340: 230-233Crossref PubMed Scopus (770) Google Scholar, 40Biel M. Ruth P. Bosse E. Hullin R. Stuhmer W. Flockerzi V. Hofmann F. FEBS Lett. 1990; 269: 409-412Crossref PubMed Scopus (195) Google Scholar, 41Koch W.J. Ellinor P.T. Schwartz A. J. Biol. Chem. 1990; 265: 17786-17791Abstract Full Text PDF PubMed Google Scholar) (the total length of the cytosolic part of the NT region of α1C is ∼154 aa in the long-NT α1C). Studies of the molecular mechanism of PKC modulation of the rabbit long-NT isoform, expressed in Xenopus oocytes, identified the first 46 aa as crucial for PKC modulation (42Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). This was further narrowed down to the first 5 aa being essential for PKC action (43Shistik E. Keren-Raifman T. Idelson G.H. Blumenstein Y. Dascal N. Ivanina T. J. Biol. Chem. 1999; 274: 31145-31149Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Recently, similar N-terminal isoforms of α1C have also been discovered in the human L-VDCC. The novel exon of the human α1C gene, exon 1a, encodes a 46-aa section at the beginning of the N terminus of α1C (the human long-NT isoform), highly homologous to the rabbit long-NT (44Blumenstein Y. Kanevsky N. Sahar G. Barzilai R. Ivanina T. Dascal N. J. Biol. Chem. 2002; 277: 3419-3423Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 45Dai B. Saada N. Echetebu C. Dettbarn C. Palade P. Biochem. Biophys. Res. Commun. 2002; 296: 429-433Crossref PubMed Scopus (36) Google Scholar). The previously known isoform, human short-NT, contains exon 1b at the beginning of the N terminus, which encodes a section of 16 aa (46Soldatov N.M. Genomics. 1994; 22: 77-87Crossref PubMed Scopus (132) Google Scholar). This isoform, used by Bouron et al. (36Bouron A. Soldatov N.M. Reuter H. FEBS Lett. 1995; 377: 159-162Crossref PubMed Scopus (37) Google Scholar) and Oz et al., (37Oz M. Melia M.T. Soldatov N.M. Abernethy D.R. Morad M. Mol. Pharmacol. 1998; 54: 1106-1112Crossref PubMed Scopus (46) Google Scholar), is not up-regulated by PKC, probably because it does not contain the segment crucial for the PKC-induced enhancement of L-VDCC. The long-NT isoform of human L-VDCC, which does contain the crucial segment, is enhanced by PMA (44Blumenstein Y. Kanevsky N. Sahar G. Barzilai R. Ivanina T. Dascal N. J. Biol. Chem. 2002; 277: 3419-3423Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). We hypothesized that it would be possible to reconstitute the enhancing effect of AngII and ACh on L-VDCC in a heterologous expression system using the long-NT isoform of α1C. Such reconstitution may greatly facilitate further studies of molecular mechanisms of L-VDCC modulation by neurotransmitters. Here, we demonstrate the reconstitution of the enhancing effect of Gq-coupled receptors on L-VDCC in Xenopus oocytes. The pharmacological characteristics of this modulation are presented. The initial segment of the N terminus is crucial for AngII- and ACh-induced enhancement of the Ca2+ channel current. The activation of Gq, and the subsequent activation of PKC, are clearly involved. The long human isoform of α1C is modulated in a manner similar to the long rabbit isoform, whereas the human short isoform yields currents that are only inhibited by AngII and ACh. Oocyte Culture—Xenopus laevis frogs were maintained and dissected as described (47Dascal N. Lotan I. Protocols in Molecular Neurobiology. 13. Humana Press, Totowa, NJ1992: 205-225Google Scholar). Oocytes were injected with equal amounts (by weight; 2.5 or 1 ng) of the mRNAs of α1C or its mutants with α2/δ, with or without β2A, with or without 1 ng of m1R or 5 ng of AT1R or AT1RM5, and incubated for 3–5 days at 20–22 °C in NDE96 solution (96 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 2.5 mm sodium pyruvate, 50 μg/ml gentamycin, 5 mm HEPES, pH 7.5). Electrophysiology—Whole cell currents were recorded using the Gene Clamp 500 amplifier (Axon Instruments, Foster City, CA) using the two-electrode voltage clamp technique in a solution containing 40 mm Ba(OH)2, 50 mm NaOH, 2 mm KOH, and 5 mm HEPES, titrated to pH 7.5 with methanesulfonic acid (48Shistik E. Ivanina T. Puri T. Hosey M. Dascal N. J. Physiol. (Lond). 1995; 489: 55-62Crossref Scopus (128) Google Scholar). Ca2+ currents were recorded in the same solution but with 40 mm Ca(OH)2 instead of Ba(OH)2. Stock solutions of AngII (10 mm) and ACh (1 m) were stored in 10–20-μl aliquots at -20 °C and added to the recoding solution at a final concentration of 1 and 10 μm, respectively (except for the AngII dose-response experiment). Ba2+ currents were measured by 200-ms steps to +20 mV from a holding potential of -80 mV, every 30 s. U73122, bis-indolylmaleimide (Bis), and staurosporine were prepared essentially as described (49Kanki H. Kinoshita M. Akaike A. Satoh M. Mori Y. Kaneko S. Mol. Pharmacol. 2001; 60: 989-998Crossref PubMed Scopus (58) Google Scholar, 50Sharon D. Vorobiov D. Dascal N. J. Gen. Physiol. 1997; 109: 477-490Crossref PubMed Scopus (137) Google Scholar). In brief, U73122 was dissolved in Me2SO at 20 mm and stored in 10-μl aliquots at -20 °C. Oocytes were injected with 25 nl of 600 μm U73122 and incubated in 10 μm U73122 for 30 min prior to measurements. Oocytes were injected with 50 nl of 300 μm Bis and incubated in 5 μm Bis for 2–4 h before measurement. Oocytes were incubated in 3 μm staurosporine for 2–4 h before measurement. In most experiments, all oocytes were injected with 25 nl of 50 mm BAPTA or EGTA, 30 min or 2–4 h before measurement, respectively, unless otherwise stated. All organic reagents were purchased from Sigma. cDNA Constructs and mRNA—cDNAs of α1C, α2/δ, and β2A were as described (51Singer D. Biel M. Lotan I. Flockerzi V. Hofmann F. Dascal N. Science. 1991; 253: 1553-1557Crossref PubMed Scopus (441) Google Scholar). The rabbit heart α1C mutants used here were prepared in our laboratory as described (42Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). cDNA of human short-NT isoform α1C,77 (Ref. 52Soldatov N.M. Bouron A. Reuter H. J. Biol. Chem. 1995; 270: 10540-10543Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar; GenBank™ accession number Z34815) was subcloned into pGEM-HJ vector as described (44Blumenstein Y. Kanevsky N. Sahar G. Barzilai R. Ivanina T. Dascal N. J. Biol. Chem. 2002; 277: 3419-3423Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). cDNA of human long-NT isoform was constructed in our laboratory as described (44Blumenstein Y. Kanevsky N. Sahar G. Barzilai R. Ivanina T. Dascal N. J. Biol. Chem. 2002; 277: 3419-3423Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Rat m1R cDNA is in pGEM2. Rat AT1R and AT1RM5 are in pZeo (53Doan T.N. Ali M.S. Bernstein K.E. J. Biol. Chem. 2001; 276: 20954-20958Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The RNAs were prepared using a standard procedure described previously, which ensures capping of the 5′ end of the RNA and preferential inclusion of non-capped GTP in the rest of the RNA (47Dascal N. Lotan I. Protocols in Molecular Neurobiology. 13. Humana Press, Totowa, NJ1992: 205-225Google Scholar). Statistics and Data Presentation—The data are presented as mean ± S.E., n = number of cells tested. To overcome the problem of batch-tobatch variability in current amplitudes, the results were normalized as follows; in each oocyte, IBa was normalized to the basal amplitude (measured before application of an agonist). These normalized values were averaged across all oocyte batches tested. Comparisons between two groups (e.g. control and receptor-expressing groups) were tested for statistically significant differences (p < 0.05 or better) using two-tailed unpaired t test. Comparisons of amplitudes of IBa at different times in the same group were done using paired t test. Comparison between several groups was done using one-way analysis of variance (ANOVA) followed by Tukey's tests, using the SigmaStat software (SPSS Corp.). Reconstitution of Neurotransmitter Modulation of L-type Ca2+ Channel in Xenopus Oocytes—We have previously demonstrated that the long-NT isoform of rabbit cardiac L-VDCC, expressed in Xenopus oocytes, is modulated by PKC activators as in cardiac and some smooth muscle cells; PMA caused an initial increase in Ba2+ current via the channels (IBa) followed by a decrease (42Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In an attempt to similarly reconstitute the modulation of this channel by Gq-activating neurotransmitters (AngII, ACh), we expressed the relevant receptors (AT1R and m1R or m3R) in conjunction with the subunits of rabbit cardiac L-VDCC: α1C (the long-NT isoform; Ref. 39Mikami A. Imoto K. Tanabe T. Niidome T. Mori Y. Takeshima H. Narumiya S. Numa S. Nature. 1989; 340: 230-233Crossref PubMed Scopus (770) Google Scholar), α2δ, and usually also β2A. Xenopus oocytes were injected with the designated RNAs, and Ba2+ currents were measured using the two-electrode voltage clamp technique. The m1R was selected for practical reasons; it is known to be a Gq-coupled receptor, and it proved to be well expressed in oocytes. The m3R gave similar effects (data not shown). The expression of m1R and AT1R was confirmed by measuring Cl- currents that develop following activation of the receptor (Fig. 1A). The appearance of this characteristic response is due to the activation of the Gq signaling cascade, which eventually leads to release of Ca2+ from intracellular stores and the consequent activation of Ca2+-dependent Cl- channels found in the oocytes (54Dascal N. CRC Crit. Rev. Biochem. 1987; 22: 317-387Crossref PubMed Scopus (521) Google Scholar). To avoid the development of Cl- currents while measuring IBa, oocytes were injected with 25 nl of 50 mm BAPTA or EGTA 1–2 h prior to current measurements (42Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). We did not observe any differences in the effects of AngII or ACh with either chelator; therefore, the results with BAPTA and EGTA were pooled. IBa was measured by step depolarizations to +20 mV from a holding potential of -80 mV every 30 s (Fig. 1B). After allowing the current to stabilize, agonist was applied for 5 min and then washed out. Application of either ACh or AngII in oocytes that expressed the channel alone did not cause any changes in IBa (Fig. 1C, a). In contrast, in oocytes that expressed the channel and the receptor, both ACh and AngII caused an increase in the amplitude of IBa, which reached a maximum after about 5 min. The enhancement of IBa by AngII was dose-dependent with an apparent EC50 of slightly less than 1 nm, which is similar to the known affinity range in native tissues (Fig. 1D). Following the period of increase, IBa declined within the next several minutes, normally below the initial (control) level (Fig. 1C, b and c). A similar decline also occurred in the constant presence of the agonist, when the latter has not been washed out after 5 min (data not shown). The reduction of IBa did not subside even after long periods of wash, and repetitive applications of AngII did not produce additional responses (neither increase nor decrease). The irreversibility of the decay, as well as other parameters (see below), cast doubt on its physiological relevance. The magnitude of the enhancement of the current differed depending on the type of receptor used, possibly due to differences in the efficiency of expression. Fig. 1E summarizes the results of the experiments in which the channel was expressed in full subunit combination (α1Cα2/δβ2A). Currents measured at 5 min (the peak of the increase) and at 8 min (representing the period of decline), expressed as percentage of initial IBa (measured in the same cell before application of the agonist), are shown. The increase in IBa caused by both transmitters was highly reproducible and statistically significant (p < 0.01). The decline phase was always present, and the decrease in IBa within a mere 3 min following the peak was also highly reproducible. The Initial Part of the N Terminus is Crucial for Modulation by a Gq-coupled Receptor—In previous experiments using PKC activators (PMA), the initial segment of the N terminus was shown to be crucial for the enhancement of the current (42Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 43Shistik E. Keren-Raifman T. Idelson G.H. Blumenstein Y. Dascal N. Ivanina T. J. Biol. Chem. 1999; 274: 31145-31149Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Consequently, if the effect of ACh and AngII is mediated by PKC, the initial segment of the N terminus should also be crucial for this modulation. Two different N terminus deletion mutants of α1C were used in which the first 20 or 46 aa are deleted from the N terminus (ΔN2–20 and DN2–46, respectively). Oocytes that expressed the wild-type (WT) or the mutant α1C, coexpressed with α2/δ and the m1R, were subjected to a similar protocol of step depolarizations to +20 mV. In neither mutant was the current enhanced as a result of receptor activation by ACh. On the contrary, only a decrease was observed following activation (Fig. 2, A and B). The extent of the decrease was rather similar in the two deletion mutants tested. Thus, the first 20 aa that are crucial for up-regulation of the channel by PKC are also indispensable for the enhancement caused by the Gq-activating receptor, m1R (summarized in Fig. 2C). In contrast, the reduction in the current seems to be a separate effect, independent of the presence of the first 46 aa. Transmitter-induced Modulation of Human Channel Isoforms—The up-regulation by PKC was shown to depend on the 46-aa sequence encoded in the long-NT isoform of α1C by exon 1a (44Blumenstein Y. Kanevsky N. Sahar G. Barzilai R. Ivanina T. Dascal N. J. Biol. Chem. 2002; 277: 3419-3423Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The alternative exon is 1b, which encodes 16 aa at the initial part of the N terminus in the short-NT isoform of α1C (Fig. 3A). Therefore, we expected the long-NT human isoform, and not the short-NT isoform, to be regulated in a manner similar to the rabbit long-NT isoform. This prediction was fulfilled; in oocytes expressing the short-NT isoform of human α1C along with α2δ, β2A, and m1R, there was a decrease of 18.3 ± 3% from the initial IBa at 5 min, whereas we observed a 30.8 ± 6% increase in oocytes expressing the human long-NT isoform. That is, a 49% difference in the peak current between the long and the short isoform (Fig. 3, B and C). These effects were absent in oocytes not expressing any receptor (data not shown). Activation of Gq Leads to Channel Modulation—Gq normally activates phospholipase C (PLC), and the latter leads to Ca2+ release and PKC activation. To examine whether PLC is involved in the modulation described above, we have expressed a mutated AT1 receptor in which the last five tyrosines of the C terminus were mutated to phenylalanines (AT1RM5). This mutated receptor was shown to lack Gq coupling (53Doan T.N. Ali M.S. Bernstein K.E. J. Biol. Chem. 2001; 276: 20954-20958Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Oocytes expressed the rabbit long-NT channel (in the α1Cα2/δβ2A composition) and the wild-type receptor or the mutated one. No Ca2+-dependent Cl- current response to AngII was observed in oocytes expressing AT1RM5 in the absence of Ca2+ chelators (compare Fig. 4, A and B). This result confirms the lack of coupling of AT1RM5 to PLC and demonstrates that activation of this receptor does not elevate Ca2+ in the oocytes. The enhancement in the current via L-type Ca2+ channel (rabbit long-NT α1C) following AngII application was present only in oocytes expressing the wild-type receptor, and only a decrease was observed with AT1RM5 (Fig. 4C). Peak current, measured 5 min after AngII application, was enhanced by 18.2 ± 4% in oocytes expressing the wild-type receptor but was decreased by 9.5 ± 4.7% in oocytes expressing the mutated receptor. That is, a difference of 28% (p < 0.001; Fig. 4D) was seen. As an additional test for the involvement of PLC, we used the PLC inhibitor U73122. Oocytes were both injected and incubated with U73122 prior to current measurement (see “Experimental Procedures”). Treatment with U73122 of oocytes expressing the WT channel (rabbit long-NT α1C) and either m1R or AT1R resulted in a complete abolishment of the enhancement as compared with untreated oocytes (Fig. 5, A and B). The difference between currents with and without U73122 was 56.5% with m1R and and 34.2% with AT1R (measured after 5 min; 33.3 ± 3.1% and 19.1 ± 1.2% increase in untreated oocytes versus a 23.3 ± 2.7% and 15.1 ± 2.4% decrease" @default.
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• W2072886932 date "2004-03-01" @default.
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• W2072886932 title "Modulation of Cardiac Ca2+ Channel by Gq-activating Neurotransmitters Reconstituted in Xenopus Oocytes" @default.
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• W2072886932 doi "https://doi.org/10.1074/jbc.m310196200" @default.
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