The N terminus of the cardiac L-type Ca(2+) 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 Ca(2+) 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 target site are not known. Moreover, uncertainties in the genomic sequence of the 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 demonstrate the presence of alpha(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 alpha(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.
Shistik
E
E
Department of Physiology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel.
Keren-Raifman
T
T
Idelson
G H
GH
Blumenstein
Y
Y
Dascal
N
N
Ivanina
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D020746:Calcium Channels, L-Type / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry*; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000302:isolation & purification / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D020746:Calcium Channels, L-Type; D010446:Peptide Fragments; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D011493:Protein Kinase C