Identification of residues in the N terminus of alpha 1B critical for inhibition of the voltage-dependent calcium channel by G beta gamma

To examine the role of the intracellular N terminus in the G-protein modula tion of the neuronal voltage-dependent calcium channel (VDCC) alpha 1B, we have pursued two routes of investigation. First, we made chimeric channels between alpha 1B and alpha 1C, the latter not being modulated by G beta gam ma subunits. VDCC alpha 1 subunit constructs were coexpressed with accessor y alpha 2 delta and beta 2a subunits in Xenopus oocytes and mammalian (COS- 7) cells. G-protein modulation of expressed alpha 1 subunits was induced by activation of coexpressed dopamine (D2) receptors with quinpirole in oocyt es, or by cotransfection of G beta 1 gamma 2 subunits in COS-7 cells. For t he chimeric channels, only those with the N terminus of alpha 1B showed any G-protein modulation; further addition of the first transmembrane domain a nd I-II intracellular linker of alpha 1B increased the degree of modulation . To determine the amino acids within the alpha 1B N terminus, essential fo r G-protein modulation, we made mutations of this sequence and identified t hree amino acids (S48, R52, and R54) within an 11 amino acid sequence as be ing critical for G-protein modulation, with I49 being involved to a lesser extent. This sequence may comprise an essential part of a complex G beta ga mma-binding site or be involved in its subsequent action.