Crucial role of N terminus in function of cardiac L-type Ca2+ channel and its modulation by protein kinase C

Abstract

The role of the cytosolic N terminus of the main subunit (α(1C)) of cardiac L-type voltage-dependent Ca2+ channel was studied in Xenopus oocyte expression system. Deletion of the initial 46 or 139 amino acids (a.a.) of rabbit heart α(1C) caused a 5-10-fold increase in the whole cell Ca2+ channel current carried by Ba2+ (I(Ba)), as reported previously (Wei, X., Neely, A., Olcese, R., Lang, W., Stefani, E., and Birnbaumer, L. (1996) Recept. Channels 4, 205-215). The plasma membrane content of α(1C) protein, measured immunochemically, was not altered by the 46-a.a. deletion. Patch clamp recordings in the presence of a dihydropyridine agonist showed that this deletion causes a ~10-fold increase in single channel open probability without changing channel density. Thus, the initial segment of the N terminus affects channel gating rather than expression. The increase in I(Ba) caused by coexpression of the auxiliary β(2A) subunit was substantially stronger in channels with full-length α(1C) than in 46- or 139-a.a. truncated mutants, suggesting an interaction between β(2A) and N terminus. However, only the I- II domain linker of α(1C), but not to N or C termini, bound β(2A) in vitro. The well documented increase of I(Ba) caused by activation of protein kinase C (PKC) was fully eliminated by the 46-a.a. deletion. Thus, the N terminus of α(1C) plays a crucial role in channel gating and PKC modulation. We propose that PKC and β subunit enhance the activity of the channel in part by relieving an inhibitory control exerted by the N terminus. Since PKC up- regulation of L-type Ca2+ channels has been reported in many species, we predict that isoforms of α(1C) subunits containing the initial N-terminal 46 a.a. similar to those of the rabbit heart α(1C) are widespread in cardiac and smooth muscle cells.