MU-OPIOID RECEPTOR-MEDIATED REDUCTION OF NEURONAL CALCIUM CURRENT OCCURS VIA A G(O)-TYPE GTP-BINDING PROTEIN

It has recently been shown that the activation of mu-opioid receptors inhibits several components of calcium channel current in rat DRG sens ory neurons. mu-Opioid receptors, acting through the pertussis toxin ( PTX)-sensitive substrate G(i), also reduce the activity of neuronal ad enylate cyclase, but the relationship of this effect to changes in cal cium channel activity has yet to be determined. Using whole-cell recor dings from acutely isolated rat DRG neurons, we examined the ability o f the mu-opioid-selective agonist Tyr-Pro-NMe-Phe-D-Pro-NH2 (PLO17) to reduce calcium current after treatment with PTX and in the presence o f the nonhydrolyzable GTP analog guanosine 5'-[-thio]triphosphate (GTP gamma S), to assess the role of G-proteins in the coupling of mu-opio id receptors to calcium channels. Inhibition of current by PLO17 was m imicked or rendered irreversible by intracellular administration of GT P gamma S, an activator of G-proteins, and was blocked by pretreatment of neurons with PTX. In contrast, when the catalytic subunit of cAMP- dependent protein kinase was included in the recording pipette, calciu m currents increased in magnitude throughout the recording without att enuation of responses to PLO17. Thus, the mu-opioid-induced inhibition of calcium current occurs through activation of a G(i)- or G(o)-type G-protein, but independent of changes in adenylate cyclase activity. A s a first step in identifying this G-protein, we compared the ability of several antisera directed against specific regions of G(i) and G(o alpha) subunits to block the inhibition in current by PLO17. Intracell ular dialysis with an antiserum specific for G(o) (GC/2) attenuated ca lcium current inhibition by PLO17 in five of six neurons by an average of 75%. In contrast, there was no attenuation in the response to PLO1 7 when neurons were dialyzed with an anti-G(i1 alpha)/G(i2 alpha) anti serum (AS/7) or antibodies specific for alpha subunits of G(i) protein s (G(i1)/G(i2) or G(i3)) in an identical manner. These results suggest that in rat DRG neurons mu-opioid receptors couple to calcium channel s via the PTX-sensitive G(o) subclass of GTP-binding proteins.