Opioid-activated postsynaptic, inward rectifying potassium currents in whole cell recordings in substantia gelatinosa neurons

Using tight-seal, whole cell recordings from isolated transverse slices of hamster and rat spinal cord, we investigated the effects of the mu-opioid a gonist (D-Ala(2), N-Me-Phe(4), Gly(5)-ol)-enkephalin (DAMGO) on the membran e potential and conductance of substantia gelatinosa (SG) neurons. We obser ved that bath application of 1-5 mu M DAMGO caused a robust and repeatable hyperpolarization in membrane potential (V-m) and decrease in neuronal inpu t resistance (RN) in 60% (27/45) of hamster neurons and 39% (9/23) of rat n eurons, but significantly only when ATP(2 mM)and guanosine 5'-triphosphate( GTP; 100 mu M) were included in the patch pipette internal solution. An ED 50 of 50 nM was observed for the hyperpolarization in rat SG neurons. Becau se G-protein mediation of opioid effects has been shown in other systems, w e tested if the nucleotide requirement for opioid hyperpolarization in SG n eurons was due to G-protein activation. GTP was replaced with the nonhydrol yzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S; 100 m u M), which enabled DAMGO to activate a nonreversible membrane hyperpolariz ation. Further, intracellular application of guanosine-5'-O-(2-thiodiphosph ate) (GDP-beta-S; 500 mu M), which blocks G-protein activation, abolished t he effects of DAMGO. We conclude that spinal SG neurons are particularly su sceptible to dialysis of GTP by whole cell recording techniques. Moreover, the depletion of GTP leads to the inactivation of G-proteins that mediate m u-opioid activation of an inward-rectifying, potassium conductance in these neurons. These results explain the discrepancy between the opioid-activate d hyperpolarization in SG neurons observed in previous sharp electrode expe riments and the more recent failures to observe these effects with whole ce ll patch techniques.