Crucial role of N terminus in function of cardiac L-type Ca2+ channel and its modulation by protein kinase C.
The role of the cytosolic N terminus of the main subunit (alpha1C) 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 alpha1C caused a 5-10-fold increase in the whole cell Ca2+ channel current carried by Ba2+ (IBa), 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 alpha1C 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 approximately 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 IBa caused by coexpression of the auxiliary beta2A subunit was substantially stronger in channels with full-length alpha1C than in 46- or 139-a.a. truncated mutants, suggesting an interaction between beta2A and N terminus. However, only the I-II domain linker of alpha1C, but not to N or C termini, bound beta2A in vitro. The well documented increase of IBa caused by activation of protein kinase C (PKC) was fully eliminated by the 46-a.a. deletion. Thus, the N terminus of alpha1C plays a crucial role in channel gating and PKC modulation. We propose that PKC and beta 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 alpha1C subunits containing the initial N-terminal 46 a.a. similar to those of the rabbit heart alpha1C are widespread in cardiac and smooth muscle cells.
Shistik
E
E
Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel.
Ivanina
T
T
Blumenstein
Y
Y
Dascal
N
N
D000595:Amino Acid Sequence; D000818:Animals; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D020746:Calcium Channels, L-Type; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D011994:Recombinant Proteins; D011493:Protein Kinase C