| Activation of G-protein coupled receptors (GPCRs) by neurotransmitters has been shown to induce inhibition of several types of voltage-dependent Ca2+ channels (VDCCs). At nerve terminals, this inhibition is important in modulating the strength of synaptic communication. N-type and P/Q-type Ca2+ channels are the major VDCCs located at the presynaptic nerve terminal and are involved in regulating neurotransmission, but only the N-type channels were studied here. The major mechanism underlying the GPCR inhibition of VDCCs is rapid, voltage-dependent, and mediated by direct binding of Gβγ subunits to the al subunit of Ca2+ channels. However, there are many examples of GPCR activation that do not produce VD inhibition of Ca2+ channels, although Gβγ subunits are always released upon receptor activation. In many cases these GPCRs are linked to Gq/11 family. The reasons for this specificity are not clear, but Gβ5γ2 has been shown to interact selectively with Gαq. The purpose of the present study was to understand the specificity of VDCC modulation by Gβγ released from Gq/11, family, using molecular, electrophysiological and cell imaging techniques.; Since there are multiple types of Gβ and Gγ subunits, many Gβγ combinations are possible. Initial studies were performed to test the ability of different Gβγ heterodimers to modulate N-type Ca2+ channels expressed in a stable cell line. The results indicated that all of the Gβ subunits could produce strong Ca 2+ inhibition. However, for Gβ5, the nature of the Gγ subunit involved was critical. Biochemical studies have shown that GGL-containing RGS proteins specifically interact with Gβ5. The ability of GGL-containing RGS proteins to modulate Ca2+ channel inhibition by Gβ5γ2 was studied. Electrophysiological studies demonstrated that these RGS proteins could antagonize Gβ5γ2-mediated N-type channel inhibition, and the effect required the GGL-domain. Association of GGL-containing RGS proteins with Gβ5 was confirmed in cells with fluorescence resonance energy transfer (FRET) studies. Finally, subcellular localization of Gβ5 and RGS11 proteins were studied using fluorescent and confocal microscopy. A model is proposed in which GGL-containing RGS proteins selectively modulate Gβ5γ2-mediated channel inhibition, and may partially underlie the specificity of VDCC modulation. |
