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Role of the Outer -Sheet in Divalent Cation Modulation of 7 Nicotinic Receptors

Departments of Pharmacology (J.T.M., J.F., A.D.S., R.L.R.) and Cell & Molecular Physiology (R.L.R.), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

-7 Nicotinic acetylcholine receptors (AChRs) exhibit a positive modulation by divalent cations similar to that observed in other AChRs. In the chick 7 AChR, this modulation involves a conserved glutamate in loop 9 (Glu¹⁷²) that undergoes agonist-dependent movements during activation. From these observations, we hypothesized that movements of the nearby -sheet formed by the 7, 9, and 10 strands may be involved in agonist activation and/or divalent modulation. To test this hypothesis, we examined functional properties of cysteine mutations of the 7 and 10 strands, alone or in pairs. We postulated that reduced flexibility or mobility of the 7/9/10-sheet as a result of introduction of a disulfide bond between the strands would alter activation by agonists. Using a nondesensitizing 7 mutant background (L²⁴⁷T), we identified one mutant pair, K¹⁴⁴C + T¹⁹⁸C, that exhibited a unique characteristic: it was fully activated by divalent cations (Ca²⁺, Ba²⁺, or Sr²⁺) in the absence of acetylcholine (ACh). Divalent-evoked currents were blocked by the 7 antagonist methyllycaconitine and were abolished when Glu¹⁷² was mutated to glutamine. When the K¹⁴⁴C + T¹⁹⁸C pair was expressed in wild-type 7 receptors, activation required both ACh and divalent cations. We conclude that the introduction of a disulfide bond into 7/9/10 lowers the energetic barrier between open and closed conformations, probably by reducing the torsional flexibility of the -sheet. In this setting, divalent cations, acting at the conserved glutamate in loop 9, act as full agonists or requisite coagonists.

Address correspondence to: Dr. James T. McLaughlin, Department of Pharmacology, CB# 7365, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365. E-mail: jmclaughlin{at}unc.edu

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