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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.

Received for publication February 7, 2006.

Accepted for publication March 13, 2006.

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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