Both Gi and Go Heterotrimeric G Proteins Are Required to Exert the Full Effect of Norepinephrine on the β-Cell KATP Channel

Geoffrey W.G Sharp,Susanne G Straub,Qinghua Fang,Ying Zhao

doi:10.1074/jbc.m707695200

Geoffrey W.G Sharp, Susanne G Straub + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m707695200

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Abstract

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.

Highlights

Two important physiological inhibitors of insulin secretion, activate Gi and/or Go proteins and inhibit the exocytosis of insulin-containing granules [2]
Considering the effects of catecholamines on the membrane potential in the insulin-secreting cells, it is clear that catecholamines can repolarize the cell membrane in a pertussis toxin (PTX)-sensitive manner
Islet cells from these mice are hyperpolarized by epinephrine in a PTX-sensitive manner, and the hyperpolarization is due to activation of a low conductance Kϩ channel [6]

Summary

EXPERIMENTAL PROCEDURES

For the standard whole cell configuration, the patch pipette was filled with solution C containing 145 mM potassium glutamate, 8 mM NaCl, 1.0 mM MgCl2, 2 mM ATP-Mg, 0.5 mM GTP, 0.3 mM cAMP, and 10 mM HEPES-KOH (pH 7.3). The outside-out patch was established after the formation of standard whole cell configuration and was continuously bathed in solution A supplemented with 5 mM tetraethylammonium and 2.8 mM glucose. The pipette solution specific for the single channel recording (solution D) was composed of 140 mM KCl, 8 mM NaCl, 1.0 mM MgCl2, 0.1 mM ATP-Mg, 0.5 mM GTP, 0.3 mM cAMP, 5 mM EGTA, and 10 mM HEPES-KOH (pH 7.3). In order to reach equilibrium of the antibodies between the pipette tip and the cytosol, monitoring the membrane potential as well as establishing the outside-out membrane patch were performed 2–5 min after the cell membrane was ruptured.

RESULTS

ATP was omitted from the pipette

DISCUSSION

Gi and