Both Gi and Go Heterotrimeric G proteins are required to exert the full effect of norepinephrine on the β-cell KATP channel

The effects of norepinephrine (NE), an inhibitor of insulin secretion, were examined on membrane potential and the ATP-sensitive K+ channel (K-ATP) in INS 832/13 cells. Membrane potential was monitored under the whole cell current clamp mode. NE hyperpolarized the cell membrane, an effect that was abolished by tolbutamide. The effect of NE on K-ATP channels was investigated in parallel using outside-out single channel recording. This revealed that NE enhanced the open activities of the K-ATP channels similar to 2-fold without changing the single channel conductance, demonstrating that NE-induced hyperpolarization was mediated by activation of the K-ATP channels. The NE effect was abolished in cells preincubated with pertussis toxin, indicating coupling to heterotrimeric G(i)/G(o) proteins. To identify the G proteins involved, antisera raised against alpha and beta subunits (anti-G alpha(common), anti-G beta, anti-G alpha(i1/2/3), and anti-G alpha(o)) were used. Anti-G alpha(common) totally blocked the effects of NE on membrane potential and KATP channels. Individually, anti-G alpha(i1/2/3) and anti-G alpha(o) only partially inhibited the action of NE on K-ATP channels. However, the combination of both completely eliminated the action. Antibodies against G(beta) had no effects. To confirm these results and to further identify the G protein subunits involved, the blocking effects of peptides containing the sequence of 11 amino acids at the C termini of the alpha subunits were used. The data obtained were similar to those derived from the antibody work with the additional information that G alpha(i3) and G alpha(o1) were not involved. In conclusion, both G(i) and G(o) proteins are required for the full effect of norepinephrine to activate the K-ATP channel.