Differential interactions of the C terminus and the cytoplasmic I-II loop of neuronal Ca2+ channels with G-protein alpha and beta gamma subunits. II. Evidence for direct binding.
The present study was designed to obtain evidence for direct interactions of G-protein alpha (Galpha) and beta gamma subunits (Gbeta gamma) with N- (alpha1B) and P/Q-type (alpha1A) Ca2+ channels, using synthetic peptides and fusion proteins derived from loop 1 (cytoplasmic loop between repeat I and II) and the C terminus of these channels. For N-type, prepulse facilitation as mediated by Gbeta gamma was impaired when a synthetic loop 1 peptide was applied intracellularly. Receptor agonist-induced inhibition of N-type as mediated by Galpha was also impaired by the loop 1 peptide but only when applied in combination with a C-terminal peptide. For P/Q-type channels, by contrast, the Galpha-mediated inhibition was diminished by application of a C-terminal peptide alone. Moreover, in vitro binding analysis for N- and P/Q-type channels revealed direct interaction of Galpha with C-terminal fusion proteins as well as direct interaction of Gbeta gamma with loop 1 fusion proteins. These findings define loop 1 of N- and P/Q-type Ca2+ channels as an interaction site for Gbeta gamma and the C termini for Galpha.
Furukawa
T
T
Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156, Japan.
Miura
R
R
Mori
Y
Y
Strobeck
M
M
Suzuki
K
K
Ogihara
Y
Y
Asano
T
T
Morishita
R
R
Hashii
M
M
Higashida
H
H
Yoshii
M
M
Nukada
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002120:Calcium Channel Agonists / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014981:Xenopus
D002120:Calcium Channel Agonists; D015220:Calcium Channels; D017931:DNA Primers; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins