QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: mol.108.050500v1 mol.108.050500v2 74/6/1687    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stewart, D. Articles by Forman, S. A. Search for Related Content PubMed PubMed Citation Articles by Stewart, D. Articles by Forman, S. A.

Tryptophan Mutations at Azi-Etomidate Photo-Incorporation Sites on ₁ or β₂ Subunits Enhance GABA_A Receptor Gating and Reduce Etomidate Modulation

Beecher-Mallincrodt Laboratories, Department of Anesthesia & Critical Care, Massachusetts General Hospital, Boston, Massachusetts

The potent general anesthetic etomidate produces its effects by enhancing GABA_A receptor activation. Its photolabel analog [³H]azi-etomidate labels residues within transmembrane domains on and β subunits: Met236 and βMet286. We hypothesized that these methionines contribute to etomidate sites formed at -β subunit interfaces and that increasing side-chain bulk and hydrophobicity at either locus would mimic etomidate binding and block etomidate effects. Channel activity was electrophysiologically quantified in ₁β_22L receptors with ₁M236W or β₂M286W mutations, in both the absence and the presence of etomidate. Measurements included spontaneous activation, GABA EC₅₀, etomidate agonist potentiation, etomidate direct activation, and rapid macrocurrent kinetics. Both ₁M236W and β₂M286W mutations induced spontaneous channel opening, lowered GABA EC₅₀, increased maximal GABA efficacy, and slowed current deactivation, mimicking effects of etomidate on ₁β_22L channels. These changes were larger with ₁M236W than with β₂M286W. Etomidate (3.2 µM) reduced GABA EC₅₀ much less in ₁M236Wβ_22L receptors (2-fold) than in wild type (23-fold). However, etomidate was more potent and efficacious in directly activating ₁M236Wβ_22L compared with wild type. In ₁β₂M286W_2L receptors, etomidate induced neither agonist-potentiation nor direct channel activation. These results support the hypothesis that ₁Met236 and β₂Met286 are within etomidate sites that allosterically link to channel gating. Although ₁M236W produced the larger impact on channel gating, β₂M286W produced more profound changes in etomidate sensitivity, suggesting a dominant role in drug binding. Furthermore, quantitative mechanistic analysis demonstrated that wild-type and mutant results are consistent with the presence of only one class of etomidate sites mediating both agonist potentiation and direct activation.

Received for publication July 14, 2008.

Accepted for publication September 18, 2008.

Address correspondence to: Dr. Stuart A. Forman, Beecher/Mallincrodt Labs, Dept. of Anesthesia and Critical Care, Gray-Jackson 4, Mass. General Hospital, 55 Fruit Street, Boston, MA 02114. E-mail: saforman{at}partners.org

This article has been cited by other articles:

				G.-D. Li, D. C. Chiara, J. B. Cohen, and R. W. Olsen Neurosteroids Allosterically Modulate Binding of the Anesthetic Etomidate to {gamma}-Aminobutyric Acid Type A Receptors J. Biol. Chem., May 1, 2009; 284(18): 11771 - 11775. [Abstract] [Full Text] [PDF]