Identification of Residues in the N Terminus of α1B Critical for Inhibition of the Voltage-Dependent Calcium Channel by Gβγ

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Abstract
To examine the role of the intracellular N terminus in the G-protein modulation of the neuronal voltage-dependent calcium channel (VDCC) α1B, we have pursued two routes of investigation. First, we made chimeric channels between α1B and α1C, the latter not being modulated by Gβγ subunits. VDCC α1 subunit constructs were coexpressed with accessory α2δ and β2a subunits inXenopus oocytes and mammalian (COS-7) cells. G-protein modulation of expressed α1 subunits was induced by activation of coexpressed dopamine (D2) receptors with quinpirole in oocytes, or by cotransfection of Gβ1γ2 subunits in COS-7 cells. For the chimeric channels, only those with the N terminus of α1B showed any G-protein modulation; further addition of the first transmembrane domain and I-II intracellular linker of α1B increased the degree of modulation. To determine the amino acids within the α1B 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 Gβγ-binding site or be involved in its subsequent action.