Activation and calcium potentiation of alpha7 receptors: structure and mechanism.

S SINE,; BOUZAT C; CHEN L

Cambridge

Congreso; Nicotinic Acetylcholine Receptors. Wellcome Trust.; 2014

wellcome Trust

Activation and calcium potentiation of 7 receptors: structure and mechanism. Steven M. Sine1, Cecilia Bouzat2 and Lin Chen3.1Mayo Clinic College of Medicine, Rochester, MN; 2Universidad Nacional del Sur, Bahia Blanca, Argentina; 3University of Southern California, Los Angeles, CA The 7 acetylcholine receptor is a homopentamer with five ligand binding sites, yet the functional significance of having five binding sites is unknown. To address this question, we combined single channel patch clamp recording with x-ray crystallography. To correlate7 function withACh occupancy, we recorded ACh-elicited channel openings from receptors in which four of the five sites were disabled by substitution of Thr for Tyr188. Unexpectedly, open channel lifetime is indistinguishable between receptors with four disabled binding sites and receptors with five intact sites. High-resolution crystal structures of an7/AChBP ligand binding domain chimera with bound barium, a calcium surrogate, reveal that barium ions occupy two of the five ligand binding sites. These findings suggest calcium acts as a co-agonist, binding at two sites while ACh binds to one or more of the remaining sites,thus potentiating agonist responses.To test the co-agonist hypothesis, we recorded ACh-elicited single channel currents in the presence and absence of divalent cations. At limiting low concentrations of ACh,7 channel openings were approximately ten-fold brieferin the absence of divalent cationsthan in the presence of calcium or barium, demonstrating strong potentiation. In receptors with four disabled ligand binding sites, ACh-elicited channel openings were again much briefer in the absence of calcium than in its presence, reconciling our initial observation, made in the presence of calcium, that open channel lifetime was independent of agonist occupancy. The overall findings demonstrate that calcium acts as a co-agonist of7, potentiating the response to agonist, and enabling activation by low concentrations of ACh prevalent in volume transmission where micron-scale distances separate 7 from ACh-containing nerve terminals.