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Agonist-Induced Conformational Changes in the Extracellular Domain of 7 Nicotinic Acetylcholine Receptors

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

The molecular mechanisms that couple agonist binding to the gating of Cys-loop ionotropic receptors are not well understood. The crystal structure of the acetylcholine (ACh) binding protein has provided insights into the structure of the extracellular domain of nicotinic receptors and a framework for testing mechanisms of activation. Key ligand binding residues are located at the C-terminal end of the 9 strand. At the N-terminal end of this strand (loop 9) is a conserved glutamate [E¹⁷² in chick 7 nicotinic acetylcholine receptors (nAChRs)] that is important for modulating activation. We hypothesize that agonist binding induces the movement of loop 9. To test this, we used the substituted-cysteine accessibility method to examine agonist-dependent changes in the modification of cysteines introduced in loop 9 of L²⁴⁷T 7 nAChRs. In the absence of agonist, ACh-evoked responses of E¹⁷²C/L²⁴⁷T 7 nAChRs were inhibited by 2-trimethylammonioethylmethane thiosulfonate (MTSET). Agonist coapplication with MTSET reduced the extent and rate of modification. The dose-dependence of ACh activation was nearly identical with that of ACh-dependent protection from modification. ACh increased the inhibition by methanethiosulfonate reagents of N¹⁷⁰C and did not change inhibition of G¹⁷¹C receptors. The antagonist dihydro--erythroidine did not mimic the effects of ACh. Combined with a structural model, the data suggest that receptor activation includes subunit rotation and/or intrasubunit conformational changes that move N¹⁷⁰ to a more accessible position and E¹⁷² to a more protected position away from the vestibule. Thus, loop 9, located near the junction between the extracellular and transmembrane domains, participates in conformational changes triggered by ligand binding.

Address correspondence to: Dr. Robert L. Rosenberg, Department of Pharmacology, CB #7365, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365. E-mail: bobr{at}med.unc.edu

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