The N terminus of the Cardiac L-type Ca2+ Channel α1C Subunit

The first 46 amino acids (aa) of the N terminus of the rabbit heart (RH) L-type cardiac Ca2+ channel α1C subunit are crucial for the stimulating action of protein kinase C (PKC) and also hinder channel gating (Shistik, E., Ivanina, T., Blumenstein, Y., and Dascal, N. (1998) J. Biol. Chem. 273, 17901–17909). The mechanism of PKC action and the location of the PKC target site are not known. Moreover, uncertainties in the genomic sequence of the N-terminal region of α1Cleave open the question of the presence of RH-type N terminus in L-type channels in mammalian tissues. Here, we demonstrate the presence of α1C protein containing an RH-type initial N-terminal segment in rat heart and brain by using a newly prepared polyclonal antibody. Using deletion mutants of α1C expressed inXenopus oocytes, we further narrowed down the part of the N terminus crucial for both inhibitory gating and for PKC effect to the first 20 amino acid residues, and we identify the first 5 aa as an important determinant of PKC action and of N-terminal effect on gating. The absence of serines and threonines in the first 5 aa and the absence of phosphorylation by PKC of a glutathioneS-transferase-fusion protein containing the initial segment suggest that the effect of PKC does not arise through a direct phosphorylation of this segment. We propose that PKC acts by attenuating the inhibitory action of the N terminus via phosphorylation of a remote site, in the channel or in an auxiliary protein, that interacts with the initial segment of the N terminus. The first 46 amino acids (aa) of the N terminus of the rabbit heart (RH) L-type cardiac Ca2+ channel α1C subunit are crucial for the stimulating action of protein kinase C (PKC) and also hinder channel gating (Shistik, E., Ivanina, T., Blumenstein, Y., and Dascal, N. (1998) J. Biol. Chem. 273, 17901–17909). The mechanism of PKC action and the location of the PKC target site are not known. Moreover, uncertainties in the genomic sequence of the N-terminal region of α1Cleave open the question of the presence of RH-type N terminus in L-type channels in mammalian tissues. Here, we demonstrate the presence of α1C protein containing an RH-type initial N-terminal segment in rat heart and brain by using a newly prepared polyclonal antibody. Using deletion mutants of α1C expressed inXenopus oocytes, we further narrowed down the part of the N terminus crucial for both inhibitory gating and for PKC effect to the first 20 amino acid residues, and we identify the first 5 aa as an important determinant of PKC action and of N-terminal effect on gating. The absence of serines and threonines in the first 5 aa and the absence of phosphorylation by PKC of a glutathioneS-transferase-fusion protein containing the initial segment suggest that the effect of PKC does not arise through a direct phosphorylation of this segment. We propose that PKC acts by attenuating the inhibitory action of the N terminus via phosphorylation of a remote site, in the channel or in an auxiliary protein, that interacts with the initial segment of the N terminus. Voltage-dependent L-type Ca2+ channels regulate contraction of cardiac and smooth muscle and excitability and gene expression in the brain (2Snutch T.P. Reiner P.B. Curr. Opin. Neurobiol. 1992; 2: 247-253Crossref PubMed Scopus (251) Google Scholar, 3Reuter H. Nature. 1983; 301: 569-574Crossref PubMed Scopus (860) Google Scholar, 4Finkbeiner S. Greenberg M.E. J. Neurobiol. 1998; 37: 171-189Crossref PubMed Scopus (181) Google Scholar). They consist of three subunits: α1 (main, pore-forming subunit), β, and α2/δ. The α1 subunits in the heart, smooth muscle, and brain are products of the α1C gene (5De Waard M. Gurnett C.A. Campbell K.P. Ion Channels. 1996; 4: 41-87Crossref PubMed Scopus (142) Google Scholar). The existence of several cDNA isoforms and the genomic sequence of the α1C DNA suggest the presence of splice variants of RNA and thus of several isoforms of the α1Cprotein (6Koch W.J. Ellinor P.T. Schwartz A. J. Biol. Chem. 1990; 265: 17786-17791Abstract Full Text PDF PubMed Google Scholar, 7Snutch T.P. Tomlinson W.J. Leonard J.P. Gilbert M.M. Neuron. 1991; 7: 45-57Abstract Full Text PDF PubMed Scopus (294) Google Scholar, 8Soldatov N.M. Genomics. 1994; 22: 77-87Crossref PubMed Scopus (130) Google Scholar), but the actual composition of α1C protein isoforms in tissues is still poorly characterized. The α1C subunit appears to be the main target for modulation by protein kinases A and C (PKA and PKC, 1The abbreviations used are:PKCprotein kinase Caaamino acidGSTglutathione S-transferasePCRpolymerase chain reactionPKAprotein kinase APMA4-β-phorbol 12-myristate 13-acetateRHrabbit heartWTwild-typeCHAPS3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid1The abbreviations used are:PKCprotein kinase Caaamino acidGSTglutathione S-transferasePCRpolymerase chain reactionPKAprotein kinase APMA4-β-phorbol 12-myristate 13-acetateRHrabbit heartWTwild-typeCHAPS3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid respectively), although β is also a substrate (9Puri T.S. Gerhardstein B.L. Zhao X.L. Ladner M.B. Hosey M.M. Biochemistry. 1997; 36: 9605-9615Crossref PubMed Scopus (110) Google Scholar). Both kinases increase the activity of the channel (10Trautwein W. Hescheler J. Annu. Rev. Physiol. 1990; 52: 257-274Crossref PubMed Scopus (317) Google Scholar, 11Tsien R.W. Annu. Rev. Physiol. 1983; 45: 341-358Crossref PubMed Scopus (452) Google Scholar, 12Wickman K. Clapham D.E. Physiol. Rev. 1995; 75: 865-885Crossref PubMed Scopus (347) Google Scholar). PKC has been proposed to mediate the enhancement of L-type Ca2+ channels by intracellular ATP (13McHugh D. Beech D.J. J. Physiol. Lond. 1997; 500: 311-317Crossref Scopus (15) Google Scholar), angiotensin II (14Dosemeci A. Dhallan R.S. Cohen N.M. Lederer W.J. Rogers T.B. Circ. Res. 1988; 62: 347-357Crossref PubMed Scopus (215) Google Scholar), glucocorticoids (15Kato H. Hayashi T. Koshino Y. Kutsumi Y. Nakai T. Miyabo S. Biochem. Biophys. Res. Commun. 1992; 188: 934-941Crossref PubMed Scopus (32) Google Scholar), PACAP (16Chik C.L. Li B. Ogiwara T. Ho A.K. Karpinski E. FASEB J. 1996; 10: 1310-1317Crossref PubMed Scopus (48) Google Scholar), and arginine-vasopressin (17Zhang S. Hirano Y. Hiraoka M. Circ. Res. 1995; 76: 592-599Crossref PubMed Scopus (28) Google Scholar). After the initial enhancement by PKC-activating phorbol esters, the Ca2+ current is often decreased (18Lacerda A.E. Rampe D. Brown A.M. Nature. 1988; 335: 249-251Crossref PubMed Scopus (211) Google Scholar, 19Tseng G.N. Boyden P.A. Am. J. Physiol. 1991; 261: H364-H379Crossref PubMed Google Scholar), but it is not clear whether the inhibition is phosphorylation-related (20Asai T. Shuba L.M. Pelzer D.J. McDonald T.F. Am. J. Physiol. 1996; 270: H620-H627PubMed Google Scholar, 21Bourinet E. Fournier F. Lory P. Charnet P. Nargeot J. Pfluegers Arch. 1992; 421: 247-255Crossref PubMed Scopus (67) Google Scholar). The dual effect of PKC activators is fully reconstituted inXenopus oocytes expressing α1C, with or without α2/δ and/or β; the presence of β attenuates the enhancing action of PKC (21Bourinet E. Fournier F. Lory P. Charnet P. Nargeot J. Pfluegers Arch. 1992; 421: 247-255Crossref PubMed Scopus (67) Google Scholar, 22Singer-Lahat D. Gershon E. Lotan I. Hullin R. Biel M. Flockerzi V. Hofmann F. Dascal N. FEBS Lett. 1992; 306: 113-118Crossref PubMed Scopus (67) Google Scholar). In the nerve cells, either stimulation (23Yang J. Tsien R.W. Neuron. 1993; 10: 127-136Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 24Kleppisch T. Klinz F.J. Hescheler J. Brain Res. 1992; 591: 283-288Crossref PubMed Scopus (29) Google Scholar, 25Herman M.D. Reuveny E. Narahashi T. J. Physiol. ( Lond ). 1993; 462: 645-660Crossref PubMed Scopus (32) Google Scholar, 26Hall K.E. Browning M.D. Dudek E.M. Macdonald R.L. J. Neurosci. 1995; 15: 6069-6076Crossref PubMed Google Scholar) or inhibition (27Hsu K.S. Huang C.C. Kan W.M. Gean P.W. Am. J. Physiol. 1996; 271: C1269-C1277Crossref PubMed Google Scholar, 28ffrench-Mullen J.M. J. Neurosci. 1995; 15: 903-911Crossref PubMed Google Scholar, 29Doerner D. Pitler T.A. Alger B.E. J. Neurosci. 1988; 8: 4069-4078Crossref PubMed Google Scholar) of L-type channels by PKC has been reported. protein kinase C amino acid glutathione S-transferase polymerase chain reaction protein kinase A 4-β-phorbol 12-myristate 13-acetate rabbit heart wild-type 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid protein kinase C amino acid glutathione S-transferase polymerase chain reaction protein kinase A 4-β-phorbol 12-myristate 13-acetate rabbit heart wild-type 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid The α1 subunit is composed of four homologous membrane-spanning internal domains, each with six transmembrane α-helixes and a pore-forming reentrant P loop (30Catterall W.A. Trends Neurosci. 1993; 16: 500-506Abstract Full Text PDF PubMed Scopus (151) Google Scholar). C and N termini and linkers between domains I-II, II-III, and III-IV are cytosolic. The initial 46 aa of the N terminus of rabbit heart (RH) α1Care crucial for PKC modulation (1Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The cytosolic N-terminal part of RH α1C is 154 aa long. Deletion of the first 40 aa or more causes a 5–10-fold increase in the current via RH-type Ca2+ channels expressed in Xenopus oocytes (1Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar,31Wei X. Neely A. Olcese R. Lang W. Stefani E. Birnbaumer L. Recept. Channels. 1996; 4: 205-215PubMed Google Scholar). This is a result of a change in channel gating because the truncation causes an increase in open probability, without increasing the amount of α1C protein in the plasma membrane (1Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). These and additional findings led us to propose that the N terminus of α1C acts as an inhibitory gate, and its removal enhances channel activation; PKC increases the current by attenuating the inhibitory action of the N terminus (1Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). It is not known whether PKC phosphorylates the N terminus. Despite the importance of the first 46 aa of the RH-type N terminus, its presence in L-type channel proteins in vivo remains uncertain. The only other cDNA of α1C containing a stretch encoding this protein sequence is that cloned from rat aorta and heart (6Koch W.J. Ellinor P.T. Schwartz A. J. Biol. Chem. 1990; 265: 17786-17791Abstract Full Text PDF PubMed Google Scholar). α1C cDNAs cloned from rabbit lung, human heart, and rat brain (7Snutch T.P. Tomlinson W.J. Leonard J.P. Gilbert M.M. Neuron. 1991; 7: 45-57Abstract Full Text PDF PubMed Scopus (294) Google Scholar, 32Schultz D. Mikala G. Yatani A. Engle D.B. Iles D.E. Segers B. Sinke R.J. Weghuis D.O. Klockner U. Wakamori M. Wang J.J. Melvin D. Varadi G. Schwartz A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6228-6232Crossref PubMed Scopus (123) Google Scholar, 33Bouron A. Soldatov N.M. Reuter H. FEBS Lett. 1995; 377: 159-162Crossref PubMed Scopus (37) Google Scholar, 34Biel M. Ruth P. Bosse E. Hullin R. Stuhmer W. Flockerzi V. Hofmann F. FEBS Lett. 1990; 269: 409-412Crossref PubMed Scopus (193) Google Scholar) do not encode this stretch (see Fig. 2 A). It has been proposed that these variations correspond to splice variants of the α1C gene (7Snutch T.P. Tomlinson W.J. Leonard J.P. Gilbert M.M. Neuron. 1991; 7: 45-57Abstract Full Text PDF PubMed Scopus (294) Google Scholar), but even this is not certain. The structure of the genomic DNA of human α1C has not been fully resolved in this region; none of the known exons correspond to the RH-type N terminus (8Soldatov N.M. Genomics. 1994; 22: 77-87Crossref PubMed Scopus (130) Google Scholar). In contrast, a recent study that utilized an RNase protection assay showed that RNA of the RH-type initial segment is predominant in human heart (35Yang Y. Chen X. Houser S.P. Biophys. J. 1999; 76: A431Google Scholar). These discrepancies make it important to clarify whether L-type Ca2+ channel isoforms with the RH-type N terminus are common in mammalian tissues. Here we demonstrate the abundance of the RH-type N terminus in rat heart and brain and map the segment critical for PKC modulation and for inhibition of gating to the very beginning of the N terminus. Our results strongly suggest that PKC effect is not mediated by phosphorylation of this initial segment. cDNAs and RNAs of RH α1C and α2/δ were as described (1Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). To create α1C N-terminal truncations, PCR amplification with Vent polymerase (New England Biolabs) was performed for α1CΔN2–5, α1CΔN2–20, and α1CΔN2–25, as described for α1CΔN2–46 and α1CΔN2–139 (1Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), introducing aSalI site followed by an initiation codon (ATG) and then by the original wild-type (WT) α1C sequence starting from the desired base (amino acid numbers correspond to the RH α1C sequence (36Mikami A. Imoto K. Tanabe T. Niidome T. Mori Y. Takeshima H. Narumiya S. Numa S. Nature. 1989; 340: PubMed Scopus Google The cDNA of the proteins of the N terminus and of the loop were described (1Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The cDNAs for and the were by a PCR and and of and thus to with by and a cDNA for encoding aa of rat brain α1C (7Snutch T.P. Tomlinson W.J. Leonard J.P. Gilbert M.M. Neuron. 1991; 7: 45-57Abstract Full Text PDF PubMed Scopus (294) Google Scholar), were and of PCR products were the proteins were by followed by with and with and with or 20 In the of were and was to and and for were from or Xenopus were and as described E. Ivanina T. T. Hosey M. Dascal N. J. Physiol. ( Lond. ). 1995; PubMed Scopus Google Scholar). were with of the of α1C or its of in the in Fig. 2 of for 5 for and for in 2 5 were using the using the in a containing 40 2 and 5 to with acid E. Ivanina T. T. Hosey M. Dascal N. J. Physiol. ( Lond. ). 1995; PubMed Scopus Google Scholar). and were performed using were by a to 20 from a of To study the effect of the were (see E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google for of and were by M. M. Hosey Zhao X. T.S. D. E. Hosey M.M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). A was the protein were with of in and with of in were the The in the were by assay on in the presence of of The were for and E. were prepared using to the of the was prepared from the by and in To the was with The was with The was with the several were from To the was performed with E. The was to 2 or and with The were and then containing was performed as described E. Ivanina T. T. Hosey M. Dascal N. J. Physiol. ( Lond. ). 1995; PubMed Scopus Google Scholar). were with and in containing for 5 oocytes were and proteins were and on from were in and on in a containing the were prepared by for to the and the and then for and of the in the The amount of protein was by the The were in were on and to for with the the or The were using the The was as described by and T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). of each protein were for 20 with of in of 5 The products were in of for proteins were on an containing and to A polyclonal a protein of the first 46 aa of the RH-type α1C N terminus was in rabbit a containing the 46 aa of RH α1C to in the of was a more protein, in was to was with of the and in the of the C E. Ivanina T. T. Hosey M. Dascal N. J. Physiol. ( Lond. ). 1995; PubMed Scopus Google Scholar, Zhao X. T.S. D. E. Hosey M.M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). the RH α1C protein expressed in Xenopus oocytes and with but not the the first 46 α1CΔN2–46 and both the α1C and α1CΔN2–46 and the of expression of the α1C was that of α1CΔN2–46 as (1Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). the channel more A). α1C was in oocytes that were not with RNA by of the and The that of the of α1C are by to the N and C termini and a of the channel the E. Ivanina T. T. Hosey M. Dascal N. J. Physiol. ( Lond. ). 1995; PubMed Scopus Google Scholar) that the the protein not of its the used the α1C protein on as an as in the oocytes (1Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, E. Ivanina T. T. Hosey M. Dascal N. J. Physiol. ( Lond. ). 1995; PubMed Scopus Google Scholar). the of this protein is (36Mikami A. Imoto K. Tanabe T. Niidome T. Mori Y. Takeshima H. Narumiya S. Numa S. Nature. 1989; 340: PubMed Scopus Google Scholar), the of its is The result from the that proteins to on T. 1991; 4: Scholar). of rat with a and a of both was in the presence of the protein the was This result the of and for the first on the protein the presence of an RH-type initial segment in the N terminus of α1C in the rat of rat heart and were with three an in the A was in tissues. we the that this is because this was not by the other The and the in and in the although with was This be because of sequence between the rabbit cardiac α1C and isoforms of rat brain channel that in the loop I. S. N. Biochem. Biol. 1998; 45: Google Scholar). the of this in and in it was even in the brain in the not brain α1C was not by but by the very of the C terminus in a of brain α1C protein be or from the cardiac additional was in the by and but not by a in the These results the T. T.S. Gerhardstein B.L. Hosey M.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (151) Google Scholar) that only a of the L-type channel in the heart is the of its C terminus (see also A. M. S. Takeshima H. S. FEBS Lett. 1992; PubMed Scopus Google Scholar). The actual of the protein is because of the of the by in α1C RNA has been in but not in M. T. 1997; 22: PubMed Scopus Google Scholar), the protein be in in the The main of this part of the study is that α1Cprotein containing the RH-type N terminus is in rat heart and and the L-type Ca2+ current in these tissues be to be by The of PKC on L-type channels (23Yang J. Tsien R.W. Neuron. 1993; 10: 127-136Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 24Kleppisch T. Klinz F.J. Hescheler J. Brain Res. 1992; 591: 283-288Crossref PubMed Scopus (29) Google Scholar, 25Herman M.D. Reuveny E. Narahashi T. J. Physiol. ( Lond ). 1993; 462: 645-660Crossref PubMed Scopus (32) Google Scholar, 26Hall K.E. Browning M.D. Dudek E.M. Macdonald R.L. J. Neurosci. 1995; 15: 6069-6076Crossref PubMed Google Scholar, K.S. Huang C.C. Kan W.M. Gean P.W. Am. J. Physiol. 1996; 271: C1269-C1277Crossref PubMed Google Scholar, 28ffrench-Mullen J.M. J. Neurosci. 1995; 15: 903-911Crossref PubMed Google Scholar, 29Doerner D. Pitler T.A. Alger B.E. J. Neurosci. 1988; 8: 4069-4078Crossref PubMed Google Scholar) result from the presence of isoforms of α1C in Deletion of the first 46 aa of the α1C, are to the RH-type N terminus 2 increases the Ca2+ channel current and also the (1Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The of 40 aa not been (1Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, X. S. Lang W. H. T. E. Birnbaumer L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). To down the segment crucial for PKC we prepared three additional deletion α1CΔN2–5, α1CΔN2–20, and α1CΔN2–25, aa and channels in a RNAs of and of four deletion mutants were prepared on the in and with α2/δ oocytes of the that deletion of the first 20 aa was to a current to that by the deletion of 46 or Deletion of aa also the current but of 20 or more of the subunit of N-terminal truncation mutants α1CΔN2–46 and we proposed that part of enhancement is because of an of the N-terminal inhibition of gating (1Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Fig. 2 that the enhancement of of by of is also that of the this was the first 20 aa are crucial for the inhibitory effect of the N terminus on L-type channel gating and on its with and the first 5 aa an important Fig. 2 current 2 and of the effect of the phorbol 2 in oocytes expressing or α1C the α2/δ in the channel showed to and the Ca2+ channel current was in Fig. 2 been used in these The removal of aa the effect by more the increase was but the first 20 aa were to the first 5 aa are very and the first 20 aa are crucial for the PKC on of PKC activators and the effect of is to result from a phosphorylation (21Bourinet E. Fournier F. Lory P. Charnet P. Nargeot J. Pfluegers Arch. 1992; 421: 247-255Crossref PubMed Scopus (67) Google Scholar, 22Singer-Lahat D. Gershon E. Lotan I. Hullin R. Biel M. Flockerzi V. Hofmann F. Dascal N. FEBS Lett. 1992; 306: 113-118Crossref PubMed Scopus (67) Google Scholar). none of the first 5 aa of α1C are serines or threonines 2 it is not that PKC phosphorylates this segment. of the in the first 20 aa is a PKC site, but a site or be a target for we in phosphorylation by PKC of proteins of of the N N and we used and the proteins of the loop of RH α1C and of the N terminus of the rat brain α1C, that and were strongly were in and in and loop were not the first 20 the protein are not these other of the N terminus The of the phosphorylation of these of the N terminus is In results demonstrate the presence of α1Cprotein containing an RH-type N terminus in rat heart and we that the initial 20 amino acids are crucial both for the inhibitory gating by the N terminus, the of the N terminus with the β and for the PKC of the first 5 aa strongly the inhibitory of the N terminus and fully the PKC The between the location of crucial for these the (1Shistik E. Ivanina T. Blumenstein Y. Dascal N. J. Biol. Chem. 1998; 273: 17901-17909Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) that PKC its stimulating action by attenuating the inhibition on the channel by the N terminus. of the mechanism of PKC action is a for the Our suggest that the effect of PKC is not by a direct phosphorylation of the initial 20 aa of the N terminus. This is both by the amino acid composition of this of the first 5 aa crucial for PKC and by the absence of phosphorylation of the protein containing the initial segment. be the mechanism of PKC are PKC a site α1C is remote from the initial N-terminal segment but interacts with it or is for N-terminal effect on gating phosphorylation by PKC the is that PKC phosphorylates an auxiliary protein, either the N-terminal inhibition the phosphorylation this or attenuates the N-terminal inhibition by We Lotan and for and for the critical of the M. Hosey for the of and and T. P. for the