Heterologous expression and coupling of G protein-gated inwardly rectifying K+ channels in adult rat sympathetic neurons.

Abstract

1. G protein-gated inwardly rectifying K+ (GIRK) channels were heterologously expressed in rat superior cervical ganglion (SCG) neurons by intranuclear microinjection. The properties of GIRK channels and their coupling to native receptors were characterized using the whole-cell patch-clamp technique. 2. Following coinjection of either GIRK1-2 or GIRK1-4 cDNA, application of noradrenaline (NA) produced large inwardly rectifying K+ currents. Injection of cDNA encoding individual GIRK subunits produced only small and inconsistent NA-activated inward currents. Current arising from the native expression of GIRK channels in SCG neurons was not observed. 3. NA-mediated activation of GIRK channels was abolished by pertussis toxin (PTX) pretreatment, indicating coupling via G proteins of the Gi/Go subfamily. Conversely, vasoactive intestinal peptide (VIP) activated GIRK channel currents via a cholera toxin-sensitive pathway suggesting coupling through Galphas. Pretreatment of neurons with PTX caused a significant increase in amplitude of the VIP-mediated GIRK channel currents when compared with untreated cells. 4. Application of adenosine, prostaglandin E2 and somatostatin resulted in activation of GIRK channel currents. Activation of m1 muscarinic acetylcholine receptors (i.e. application of oxotremorine M to PTX-treated neurons) failed to elicit overt GIRK channel currents. 5. GIRK channel overexpression decreased basal Ca2+ channel facilitation significantly when compared with uninjected neurons. Furthermore, the NA-mediated inhibition of Ca2+ channels was significantly attenuated. 6. In summary, the ability to heterologously express GIRK channels in adult sympathetic neurons allows the experimental alteration of receptor-G protein-effector stoichiometry. Such studies may increase our understanding of the mechanisms underlying ion channel modulation by G proteins in a neuronal environment.