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J. Biol. Chem., Vol. 283, Issue 9, 5306-5316, February 29, 2008

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Both G_i and G_o Heterotrimeric G Proteins Are Required to Exert the Full Effect of Norepinephrine on the β-Cell K_ATP Channel^*

Ying Zhao, Qinghua Fang, Susanne G. Straub, and Geoffrey W. G. Sharp¹

From the Department of Molecular Medicine, College of Veterinary Medicine and Department of Applied Engineering and Physics, Cornell University, Ithaca, New York 14853-6401

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 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 and β subunits (anti-G_common, anti-Gβ, anti-G_i1/2/3, and anti-G_o) were used. Anti-G_common totally blocked the effects of NE on membrane potential and K_ATP channels. Individually, anti-G_i1/2/3 and anti-G_o only partially inhibited the action of NE on K_ATP 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 subunits were used. The data obtained were similar to those derived from the antibody work with the additional information that G_i3 and G_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.

Received for publication, September 13, 2007 , and in revised form, December 7, 2007.

Note Added in Proof—Since the submission of this manuscript, a related article has been published (Katsuya, D., Kakei, M., and Yada, T. (2007) Diabetes 56, 2319–2327).

^* This work was supported by National Institutes of Health Grants DK54243 and DK56737 (to G. W. G. S.) and a Career Development Award from the Juvenile Diabetes Foundation International (to S. G. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401. Tel.: 607-253-3875; Fax: 607-253-3659; E-mail: gws2{at}cornell.edu.

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