Rapid desensitization of the rat alpha7 nAChR is facilitated by the presence of a proline residue in the outer beta-sheet

Abstract

The rat alpha7 nicotinic acetylcholine receptor (nAChR) has a proline residue near the middle of the beta9 strand. The replacement of this proline residue at position 180 (P180) by either threonine (alpha7-P180T) or serine (alpha7-P180S) slowed the onset of desensitization dramatically, with half-times of ~930 and 700 ms, respectively, compared to 90 ms for the wild-type receptor. To investigate the importance of the hydroxyl group on the position 180 side-chains, the mutant receptors alpha7-P180Y and alpha7-P180F were studied and showed half-times of desensitization of 650 and 160 ms, respectively. While a position 180 side-chain OH group may contribute to the slow desensitization rates, alpha7-P180S and alpha7-P180V resulted in receptors with similar desensitization rates, suggesting that increased backbone to backbone H bonding expected in the absence of proline at position 180 would likely exert a great effect on desensitization. Single channel recordings indicated that for the alpha7-P180T receptor there was a significantly reduced closed time without any change in single channel conductance (as compared to wild-type). Kinetic simulations indicated that all changes observed for the mutant channel behaviour were reproduced by decreasing the rate of desensitization, and increasing the microscopic affinity to resting receptors. Molecular dynamics (MD) simulations on a homology model were used to provide insight into likely H bond interactions within the outer beta-sheet that occur when the P180 residue is mutated. All mutations analysed increased about twofold the predicted number of H bonds between the residue at position 180 and the backbone of the beta10 strand. Moreover, the alpha7-P180T and alpha7-P180S mutations also formed some intrastrand H bonds along the beta9 strand, although H bonding of the OH groups of the threonine or serine side-chains was predicted to be infrequent. Our results indicate that rapid desensitization of the wild-type rat alpha7 nAChR is facilitated by the presence of the proline residue within the beta9 strand.