In the heart, Ca 2ϩ current via the voltage-dependent L-type channels (dihydropyridine-sensitive) underlies the plateau of the action potential and provides calcium ions necessary for initiation of cardiac cell contraction (2). Similar channels are found in smooth muscle, where they play a major role in regulation of tonus and contraction (3, 4), and in the nervous system (5, 6). L-type channels are composed of the following three subunits: the main, pore-forming ␣ 1C , the cytosolic 2, and the ␣ 2 ␦ subunit which is mostly extracellular (5, 7-11). ␣ 1C contains four homologous membrane domains numbered I-IV, each one with six transmembrane segments and a re-entrant P-loop that forms the pore lining; N-and C-terminal domains and the linkers connecting the domains I-II, II-II, and II-IV are cytosolic (see Ref. 7 for review, and see Fig. 6A for a scheme). The C terminus was implicated in Ca 2ϩ -and voltagedependent inactivation (12-15) and modulation by protein kinase A (16 -19); linker I-II contains the binding site for the  subunit (20, 21).Cardiac and smooth muscle L-type channels are tightly regulated by hormonal and neuronal signals via G proteins and protein kinases (22,23). Protein kinase C (PKC) 1 is one of such regulators; its actions appear to be tissue-and species-specific. PKC activators, such as phorbol esters and diacylglycerols, increase Ca 2ϩ channel currents in cardiac and smooth muscle cells of various mammals (24 -33), and PKC has been implicated in mediating the stimulation of Ca 2ϩ channels by intracellular ATP (34), angiotensin II (26), glucocorticoids (28), pituitary adenylate cyclase-activating polypeptide (33), and arginine-vasopressin (32). PKC up-regulation results from changes in channel gating because it is accompanied by an increase in single channel open probability, P o (30,35,36). In many cases, a biphasic effect of PKC activators has been described, with an increase followed by a later decrease (25,27,30), and some preparations such as adult guinea pig heart cells (37, 38) respond to phorbol esters only by a decrease in Ca 2ϩ currents, an effect that may not be mediated by PKC (38). The biphasic response to PKC stimulators is fully reconstituted when expression of L-type channels in Xenopus oocytes is directed by RNA extracted from rat heart (39, 40) or cRNA of rabbit cardiac ␣ 1C subunit (39). Increase of Ca 2ϩ channel activity by phorbol esters has also been observed in a mammalian cell line (baby hamster kidney) expressing the rabbit cardiac ␣ 1C (36). The potentiation by phorbol esters of Ca 2ϩ channels expressed in the oocytes is mediated by PKC because it is mimicked by diacylglycerols and blocked by specific PKC inhibitors (39,40).Both ␣ 1C and  are substrates for PKC-catalyzed phosphorylation (Ref. 41 and references therein). ␣ 1C subunit has been recognized as the target for the Ca 2ϩ channel enhancement caused by PKC, since coexpression of the auxiliary subunits was not necessary to reproduce the effect of phorbol esters; on the contrary, coexpression of the  subunit...
Neuronal voltage-dependent Ca2؉ channels of the N (␣ 1B ) and P/Q (␣ 1A ) type are inhibited by neurotransmitters that activate G i/o G proteins; a major part of the inhibition is voltage-dependent, relieved by depolarization, and results from a direct binding of G␥ subunit of G proteins to the channel. Since cardiac and neuronal L-type (␣ 1C ) voltage-dependent Ca 2؉ channels are not modulated in this way, they are presumed to lack interaction with G␥. However, here we demonstrate that both G␥ and calmodulin directly bind to cytosolic N and C termini of the ␣ 1C subunit. Coexpression of G␥ reduces the current via the L-type channels. The inhibition depends on the presence of calmodulin, occurs at basal cellular levels of Ca 2؉ , and is eliminated by EGTA. The N and C termini of ␣ 1C appear to serve as partially independent but interacting inhibitory gates. Deletion of the N terminus or of the distal half of the C terminus eliminates the inhibitory effect of G␥. Deletion of the N terminus profoundly impairs the Ca 2؉ /calmodulin-dependent inactivation. We propose that G␥ and calmodulin regulate the L-type Ca 2؉ channel in a concerted manner via a molecular inhibitory scaffold formed by N and C termini of ␣ 1C .Voltage-dependent Ca 2ϩ channels (VDCCs) 1 are crucial for neuronal and muscular excitability (1). Mammalian VDCCs fall into several families distinguished by pharmacological and biophysical properties (L, N, P/Q, T, and R type) and the molecular identity of the main, poreϪforming subunit, ␣ 1 (2-4). The neuronal N-and P/Q-type channels, based on ␣ 1B and ␣ 1A , respectively, are crucial for neurotransmitter release (3). Ltype Ca 2ϩ channels containing the "cardiac-type" ␣ 1C subunit regulate contraction of cardiac and smooth muscle, and excitability and gene expression in the brain (2, 5, 6). The ␣ 1 subunits contain four homologous membrane domains numbered IϪIV and 5 large intracellular segments: N terminus (NT), C terminus (CT), and linkers between the domains (often called loops L 1 , L 2 , and L 3 ). There is also a large number of short intracellular linkers between transmembrane segments within each domain.Activation in all voltage-dependent channels is initiated by a voltage-driven shift in charged transmembrane elements (7). Nevertheless, the parts of the channel and the auxiliary subunits which are not exposed to the membrane electrical field may substantially modulate the gating (for reviews related to Ca 2ϩ channels, see Ref.3). In particular, VDCCs are strongly and specifically modulated by neurotransmitters acting via heterotrimeric G proteins, via actions on the cytosolic parts of the channel. Some of the modulations are mediated by G protein-triggered second messenger cascades, often via protein kinases A and C (PKA and PKC, respectively), others by a direct interaction with G protein subunits (1, 8 -14). Both PKC and PKA alter VDCC gating parameters acting via cytosolic parts of ␣ 1 or via the ancillary  subunit (15-19).Neuronal VDCCs are usually inhibited by G protein-coupled rece...
Human L-type voltage-dependent Ca2؉ channels (␣ 1C , or Ca v 1.2) are up-regulated by protein kinase C (PKC) in native tissues, but in heterologous systems this modulation is absent. In rat and rabbit, ␣ 1C has two N-terminal (NT) isoforms, long and short, with variable initial segments of 46 and 16 amino acids, respectively. The initial 46 amino acids of the long-NT ␣ 1C are crucial for PKC regulation. However, only a short-NT human ␣ 1C is known. We assumed that a long-NT isoform of human ␣ 1C may exist. By homology screening of human genomic DNA, we identified a stretch (termed exon 1a) highly homologous to rabbit long-NT, separated from the next known exon of ␣ 1C (exon 1b, which encodes the alternative, short-NT) by an ϳ80 kb-long intron. The predicted 46-amino acid protein sequence is highly homologous to rabbit long-NT. Reverse transcriptase PCR showed the presence of exon 1a transcript in human cardiac RNA. Expression of human long-NT ␣ 1C in Xenopus oocytes produced Ca 2؉ channel enhanced by a PKC activator, whereas the short-NT ␣ 1C was inhibited. The long-NT isoform may be the Ca 2؉ channel enhanced by PKCactivating transmitters in human tissues.Voltage-dependent L-type Ca 2ϩ channels are crucial for cardiac and smooth muscle contraction and hormone secretion, and they regulate gene expression in the brain (1-3). Their function is highly regulated by hormones and neurotransmitters, largely via activation of protein kinases (3,4). Regulation by PKC 1 is believed to be of substantial physiological importance, mediating all or part of the effects of several hormones and intracellular messengers (4). PKC enhances L-type Ca 2ϩ currents in diverse human tissues and cell lines: heart, neuroblastoma, T-cells, and endocrine cells (5-10). Dual modulation by PKC is often observed with activation followed by, or concomitant with, inhibition (5, 10). Similar enhancement by PKC, sometimes followed by inhibition, has been described in other mammals (11,12) and was reproduced in Xenopus oocytes expressing the cloned rabbit cardiac L-type Ca 2ϩ channels (13,14). However, expression of human L-type channels, encoded by all cDNA cloned to date, yielded Ca 2ϩ channels that were only inhibited by PKC; the enhancement could not be reconstituted (15). The reason for the inability to reproduce the PKC modulation of human L-type channels remained unknown. The main, pore-forming subunit of cardiac/smooth muscle L-type channel (␣ 1C or Ca v 1.2), also present in the brain, is the product of the ␣ 1C gene, CACNA1C (16). Several splice variants of CACNA1C are known (17,18). The resulting isoforms of human ␣ 1C protein show differential distribution in human tissues, and in failing versus normal myocardium. They play important roles in Ca 2ϩ -dependent inactivation, oxygen sensing, and drug sensitivity (18 -22). However, the genomic structure of the beginning of N-terminal region of human ␣ 1C is not entirely clear. In the two best studied mammalian species, rat and rabbit, two N-terminal isoforms of ␣ 1C cDNA are known, which mos...
The first 46 amino acids (aa) of the N terminus of the rabbit heart (RH) L-type cardiac Ca 2؉ channel ␣ 1C 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 ␣ 1C leave open the question of the presence of RH-type N terminus in Ltype channels in mammalian tissues. Here, we demonstrate the presence of ␣ 1C 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 ␣ 1C expressed in Xenopus 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 glutathione S-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 Ca 2ϩ channels regulate contraction of cardiac and smooth muscle and excitability and gene expression in the brain (2-4). They consist of three subunits: ␣ 1 (main, pore-forming subunit), , and ␣ 2 /␦. The ␣ 1 subunits in the heart, smooth muscle, and brain are products of the ␣ 1C gene (5). The existence of several cDNA isoforms and the genomic sequence of the ␣ 1C DNA suggest the presence of splice variants of RNA and thus of several isoforms of the ␣ 1C protein (6 -8), but the actual composition of ␣ 1C protein isoforms in tissues is still poorly characterized.The ␣ 1C subunit appears to be the main target for modulation by protein kinases A and C (PKA and PKC, 1 respectively), although  is also a substrate (9). Both kinases increase the activity of the channel (10 -12). PKC has been proposed to mediate the enhancement of L-type Ca 2ϩ channels by intracellular ATP (13), angiotensin II (14), glucocorticoids (15), PACAP (16), and arginine-vasopressin (17). After the initial enhancement by PKC-activating phorbol esters, the Ca 2ϩ current is often decreased (18, 19), but it is not clear whether the inhibition is phosphorylation-related (20, 21). The dual effect of PKC activators is fully reconstituted in Xenopus oocytes expressing ␣ 1C , with or without ␣ 2 /␦ and/or ; the presence of  attenuates the enhancing action of PKC (21, 22). In the nerve cells, either stimulation (23-26) or inhibition (27-29) of L-type channels by PKC has been reported.The ␣ 1 subunit is co...
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