Loop-C capping increases channel opening by increasing agonist binding and not by a long-distance energy transfer. The 11 loop-C residues (Fig. 1 B) were replaced by n = 11, 9, 7, 5, or 3 glycines. (A) Gating in the absence of ligands; the background mutations increased constitutive gating but had no effect on agonist binding (αA96H in loop A and εS450W in M4). Left, the residues in loop C (V188 to Y198); middle, open-interval duration histograms; right, example currents. (Top) WT loop C. Constructs with modified C loops produce unliganded gating activity having similar properties as the WT (Table 1). Progressive replacement with glycines hardly changes the brief component of unliganded gating, which is normal even when loop C has been deleted. (B) Activation by ACh. (Top) The 11-glycine construct was not activated by 1,000 µM ACh (compare with the 11-gly trace in A). (Bottom) With tyrosines at positions αY190 and αY198 (9-gly/2-tyr), ACh increases the open probability within clusters, indicating that the agonist can bind and increase the gating equilibrium constant (concentration in micrometer given above each trace). (C) Two point mutations in loop B of the binding site mimic the effects of loop-C cross-linking (no agonist added).