Both G i and G o heterotrimeric G proteins are required to exert the full effect of norepinephrine on the beta-cell K ATP 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 approximately 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 K ATP 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.
Zhao
Ying
Y
Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401, USA.
Fang
Qinghua
Q
Straub
Susanne G
SG
Sharp
Geoffrey W G
GW
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002460:Cell Line; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000037:antagonists & inhibitors / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology; D054086:KATP Channels / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009638:Norepinephrine / Q000494:pharmacology*; D051381:Rats; D013566:Sympathomimetics / Q000494:pharmacology*
D000906:Antibodies; D044387:GTP-Binding Protein beta Subunits; D007328:Insulin; D054086:KATP Channels; D013566:Sympathomimetics; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D009638:Norepinephrine