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G protein β Subunits Modulate the Number and Nature of Exocytotic Fusion Events in Adrenal Chromaffin Cells Independent of Calcium Entry

¹Departments of Anesthesiology and ²Pharmacology and ³Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennessee

Submitted 31 July 2008; accepted in final form 16 September 2008

G-protein-coupled receptors (GPCR) play important roles in controlling neurotransmitter and hormone release. Inhibition of voltage-gated Ca²⁺ channels (Ca²⁺ channels) by G protein β subunits (Gβ) is one prominent mechanism, but there is evidence for additional effects distinct from those on calcium entry. However, relatively few studies have investigated the Ca²⁺-channel-independent effects of Gβ on transmitter release, so the impact of this mechanism remains unclear. We used carbon fiber amperometry to analyze catecholamine release from individual vesicles in bovine adrenal chromaffin cells, a widely used neurosecretory model. To bypass the effects of Gβ on Ca²⁺ entry, we stimulated secretion using ionomycin (a Ca²⁺ ionophore) or direct intracellular application of Ca²⁺ through a patch pipette. Activation of endogenous GPCR or transient transfection with exogenous Gβ significantly reduced the number of amperometric spikes (the number of vesicular fusion events). The charge ("quantal size") and amplitude of the amperometric spikes were also significantly reduced by GPCR/Gβ. We conclude that independent from effects on calcium entry, Gβ can regulate both the number of vesicles that undergo exocytosis and the amount of catecholamine released per fusion event. We discuss possible mechanisms by which Gβ might exert these novel effects including interaction with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.