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An 7 Nicotinic Acetylcholine Receptor Gain-of-Function Mutant That Retains Pharmacological Fidelity

Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida (A.N.P., E.M.M., R.L.P.); and Istituto Pasteur-Fond. Cenci Bolognetti and Dipartimento di Fisiologia Umana e Farmacologia, Università degli Studi di Roma "La Sapienza", Rome, Italy (F.G.)

The 7-type nicotinic acetylcholine receptor (nAChR) has been recognized as a potential therapeutic target for the treatment of a variety of pathologic conditions, including schizophrenia, Alzheimer's disease, and peripheral inflammation. A unique feature of 7 nAChRs that tends to complicate functional assays intended to identify selective drugs for these receptors is the strong concentration-dependent desensitization of their agonist-evoked responses. At low agonist concentrations, voltage-clamp responses are small but tend to closely follow the solution exchange profile, whereas higher agonist concentrations produce responses that peak and then decay very rapidly, usually before the full drug concentration has been achieved. In this article, we report that an 7 T245S mutant, which has a point mutation at the sixth position in the 7 second transmembrane domain (T6'S), demonstrates a significant gain of function, sustaining current when exposed to relatively high agonist concentrations when expressed in Xenopus laevis oocytes and larger peak currents when expressed in mammalian GH4C1 cells. At the single-channel level, the T6'S mutant has a unitary conductance of 61.7 ± 5.8 pS, similar to that reported for wild-type 7, but a vastly longer average open duration. In addition, channel burst activity indicates a greater than 40% probability of channel re-opening in the sustained presence of 30 µM acetylcholine, consistent with a greater overall open probability relative to wild-type 7. Unlike the 7 L248T gain-of-function mutant, the T6'S mutant exhibits a pharmacological profile that is remarkably similar to the wild-type 7 receptor, implicating it as a potentially useful tool for identifying therapeutic agents.

Received for publication July 1, 2005.

Accepted for publication September 23, 2005.

Address correspondence to: Roger L. Papke, Department of Pharmacology and Therapeutics, P. O. Box 100267, University of Florida, Gainesville, FL 32610-0267. E-mail: rlpapke{at}ufl.edu

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