Cloning and functional characterization of squid voltage-dependent Ca2+ channel beta subunits: involvement of N-terminal sequences in differential modulation of the current.
cDNAs that encode beta subunits of voltage-dependent Ca(2+) channel were cloned from the optic lobe of the squid Loligo bleekeri. The subunits, LoCa(v)beta(1a) and LoCa(v)beta(1b) are 96% identical in amino acid sequence. The sole sequence differences are in the N-terminal region and in a five amino acid insertion in the central region of LoCa(v)beta(1b). RT-PCR revealed that LoCa(v)beta(1a) and LoCa(v)beta(1b) transcripts were expressed mainly in the optic lobe and stellate ganglion, and more weakly in mantle muscle, systemic heart, gill, branchial heart, stomach and liver. Coexpression of LoCa(v)beta(1a) or LoCa(v)beta(1b) with mammalian Ca(v)2.3 and alpha(2)/delta subunits in the Xenopus oocyte resulted in high-voltage-activated currents, and showed slow current inactivation and moderate steady-state inactivation. Comparison of the squid subunits with four mammalian beta subunits, beta(1b), beta(2a), beta(3) and beta(4), demonstrated that the modulatory effects of the beta subunits on steady-state inactivation kinetics were beta(3)<beta(4) approximately beta(1b)<LoCa(v)beta(1a) approximately LoCa(v)beta(1b)<beta(2a). LoCa(v)beta(1a)-induced current amplitude was about two to four times higher than that of LoCa(v)beta(1b). Experiments with point mutants and chimeras suggest that potential PKC and CK2 phosphorylation sites in the N-terminal region of LoCa(v)beta(1b) affect the current amplitude reciprocally, and may be responsible for regulating current amplitude.
Molecular Neurophysiology Group, Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba Central 6, Tsukuba, Ibaraki 305-8566, Japan.
Tadashi
T
Kimura
Tai
T
Kubo
D000595:Amino Acid Sequence; D000818:Animals; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D047390:Casein Kinase II; D002678:Chimera; D002999:Clone Cells; D018076:DNA, Complementary; D049832:Decapodiformes / Q000502:physiology*; D004558:Electric Stimulation; D004594:Electrophysiology; D008564:Membrane Potentials / Q000502:physiology; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D009899:Optic Lobe, Nonmammalian / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021122:Protein Subunits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D015220:Calcium Channels; D018076:DNA, Complementary; D021122:Protein Subunits; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C