Crucial Role of N Terminus in Function of Cardiac L-type Ca2+ Channel and Its Modulation by Protein Kinase C

Abstract

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...

Inhibition of cardiac L-type calcium channels by protein kinase C phosphorylation of two sites in the N-terminal domain

“…In contrast to our results, studies of a comparable cardiac Ca v 1.2 channel expressed in Xenopus oocytes showed biphasic regulation (24,25). Mutants with a truncated N terminus lost sensitivity to regulation by PKC activators (25).…”

“…However, when expressed in Xenopus oocytes, Ca v 1.2 channels typically respond to PKC activation with an initial increase in current followed by a decrease (20)(21)(22)(23)(24)(25). In contrast, we find that Ca v 1.2 channels expressed in the tsA-201 cell line of human embryonic kidney cells are inhibited by PKC, as observed in some studies of cardiac myocytes (15)(16)(17).…”

“…The function of the N terminus has been less well characterized. Similar to the C terminus, truncation greatly enhances the probability of channel opening in Xenopus oocytes and in HEK-293 cells (24,45). Our results presented here show that phosphorylation of threonine residues in the N-terminal domain by PKC can inhibit channel activity.…”

The Role of a Voltage-Dependent Ca2+Channel Intracellular Linker: A Structure-Function Analysis

“…For electrophysiology, the following cDNAs were used: rabbit Ca V 1.2 (X15539), Ca V ␤2b (L06110), ␣2␦-1 (P13806) (Shistik et al, 1998), and rabbit Ca V 2.2 ␣1(D14157). All constructs included a T7 promoter sequence for RNA transcriptions.…”

CaV1.2 I-II linker structure and Timothy syndrome

“…Xenopus maintenance and oocyte preparation were as described. 8 Oocytes were injected with RNA 3-5 d before the experiment. Due to lower channel surface expression, longer incubation time was used for oocytes not injected with Ca V β (up to 10 d).…”