The N terminus of the cardiac L-type Ca2+ channel alpha(1C) subunit - The initial segment is ubiquitous and crucial for protein kinase C modulation, but is not directly phosphorylated

The first 46 amino acids (aa) of the N terminus of the rabbit heart (RH) L- type cardiac Ca2+ channel alpha(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 targe t site are not known. Moreover, uncertainties in the genomic sequence of th e N-terminal region of alpha(1C) leave open the question of the presence of RH-type N terminus in L-type channels in mammalian tissues. Here, we demon strate the presence of alpha(1C) protein containing an RH-type initial N-te rminal segment in rat heart and brain by using a newly prepared polyclonal antibody. Using deletion mutants of alpha(1C) expressed in Xenopus oocytes, we further narrowed down the part of the N terminus crucial for both inhib itory 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 fi rst 5 aa and the absence of phosphorylation by PKC of a glutathione S-trans ferase-fusion protein containing the initial segment suggest that the effec t of PKC does not arise through a direct phosphorylation of this segment. W e propose that PKC acts by attenuating the inhibitory action of the N termi nus via phosphorylation of a remote site, in the channel or in an auxiliary protein, that interacts with the initial segment of the N terminus.