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

Abstract

The effects of norepinephrine (NE), an inhibitor of insulin secretion, were examined on membrane potential and the ATP-sensitive K+ channel (KATP) 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 KATP channels was investigated in parallel using outside-out single channel recording. This revealed that NE enhanced the open activities of the K ATP channels ∼2-fold without changing the single channel conductance, demonstrating that NE-induced hyperpolarization was mediated by activation of the KATP channels. The NE effect was abolished in cells preincubated with pertussis toxin, indicating coupling to heterotrimeric G i/Go proteins. To identify the G proteins involved, antisera raised against α and β subunits (anti- Gαcommon, anti-Gβ, anti-Gαi1/2/3, and anti-Gαo) were used. Anti-Gαcommon totally blocked the effects of NE on membrane potential and KATP channels. Individually, anti-Gαi1/2/3 and anti-Gαo only partially inhibited the action of NE on KATP channels. However, the combination of both completely eliminated the action. Antibodies against G β 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 α subunits were used. The data obtained were similar to those derived from the antibody work with the additional information that Gαi3 and Gαo1 were not involved. In conclusion, both Gi and Go proteins are required for the full effect of norepinephrine to activate the KATP channel. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.