An 7 Nicotinic Acetylcholine Receptor Gain-of-Function Mutant That Retains Pharmacological Fidelity Andon N. Placzek, Francesca Grassi, Edwin M. Meyer, and Roger L. Papke 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, Universita ` degli Studi di Roma “La Sapienza”, Rome, Italy (F.G.) Received July 1, 2005; accepted September 23, 2005 ABSTRACT 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, Alz- heimer’s disease, and peripheral inflammation. A unique feature of 7 nAChRs that tends to complicate functional assays in- tended to identify selective drugs for these receptors is the strong concentration-dependent desensitization of their ago- nist-evoked responses. At low agonist concentrations, voltage- clamp responses are small but tend to closely follow the solu- tion 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 transmem- brane domain (T6'S), demonstrates a significant gain of func- tion, 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 M 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 thera- peutic agents. Nicotinic acetylcholine receptors (nAChR) composed of the 7 subunit are pentameric, ligand-gated ion channels that are widely expressed in mammalian tissues, including both central and peripheral neurons (Dani, 2001; Skok, 2002), as well as non-neuronal cells (Wang et al., 2003; Shytle et al., 2004). Although the normal physiological functions of 7- type nAChRs are poorly understood, studies have shown behavioral effects of 7-selective agonists suggesting a role in learning and memory function (Rezvani and Levin, 2001). Other reports have implicated 7 nAChRs as potential ther- apeutic targets in schizophrenia (Freedman et al., 2000) and neurodegenerative conditions such as Alzheimer’s disease (O’Neill et al., 2002). Recent studies have also shown that 7 receptors play an important role in peripheral inflammatory processes (Wang et al., 2003), suggesting that selective tar- geting of 7 nAChRs may be beneficial in preventing the deleterious effects of septicemia. The 7-type nAChRs are unique among nicotinic receptors in that they display a high degree of concentration-depen- dence to the kinetics of agonist-evoked responses. That is, with the application of increasing concentrations of agonist, the macroscopic responses of 7 nAChRs become more and more transient, to the point where the peak of the response occurs well before the maximum concentration is achieved (Papke and Thinschmidt, 1998). This is true even for very rapid drug application systems, indicating that the macro- scopic kinetics of wild-type 7 receptors are faster than the rate of currently available agonist application devices (Papke et al., 2000a; Uteshev et al., 2002). The fact that 7 nAChRs have been reported to have a very high permeability to cal- cium (Seguela et al., 1993) suggests that this agonist concen- tration-dependent limitation of the channel’s activity may help prevent excitotoxic injury in cells with high levels of 7 receptors expressed (Li et al., 1999). Previous work in our laboratories has shown that the 6' This work was supported by National Institutes of Health grants NS043822-01A, GM57481-01A2, and PO1-AG10485. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.016402. ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; TM2, second transmembrane domain; ACh, acetylcholine; KRH, Krebs-Ringer- HEPES; 5HI, 5-hydroxyindole; P fCa , Ca 2+ fractional permeability; P open , open probability; CRC, concentration-response curve; GTS-21, (2,4)- dimethoxybenzylidene anabaseine dihydrochloride; AR-R17779, (-)-spiro[1-azabicyclo[2.2.2]octane-3,5'-oxazolidin-2'-one]. 0026-895X/05/6806-1863–1876$20.00 MOLECULAR PHARMACOLOGY Vol. 68, No. 6 Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 16402/3067618 Mol Pharmacol 68:1863–1876, 2005 Printed in U.S.A. 1863