Identification of residues in the N terminus of alpha1B critical for inhibition of the voltage-dependent calcium channel by Gbeta gamma.
To examine the role of the intracellular N terminus in the G-protein modulation of the neuronal voltage-dependent calcium channel (VDCC) alpha1B, we have pursued two routes of investigation. First, we made chimeric channels between alpha1B and alpha1C, the latter not being modulated by Gbeta gamma subunits. VDCC alpha1 subunit constructs were coexpressed with accessory alpha2delta and beta2a subunits in Xenopus oocytes and mammalian (COS-7) cells. G-protein modulation of expressed alpha1 subunits was induced by activation of coexpressed dopamine (D2) receptors with quinpirole in oocytes, or by cotransfection of Gbeta1gamma2 subunits in COS-7 cells. For the chimeric channels, only those with the N terminus of alpha1B showed any G-protein modulation; further addition of the first transmembrane domain and I-II intracellular linker of alpha1B increased the degree of modulation. To determine the amino acids within the alpha1B N terminus, essential for G-protein modulation, we made mutations of this sequence and identified three amino acids (S48, R52, and R54) within an 11 amino acid sequence as being critical for G-protein modulation, with I49 being involved to a lesser extent. This sequence may comprise an essential part of a complex Gbeta gamma-binding site or be involved in its subsequent action.
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Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom.
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D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002872:Chromosome Deletion; D018491:Dopamine Agonists / Q000494:pharmacology; D005260:Female; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000502:physiology*; D017354:Point Mutation; D017448:Receptors, Dopamine D2 / Q000819:agonists; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014981:Xenopus
D002121:Calcium Channel Blockers; D018491:Dopamine Agonists; D010446:Peptide Fragments; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins; D019204:GTP-Binding Proteins