MUTATIONS IN M2 ALTER THE SELECTIVITY OF THE MOUSE NICOTINIC ACETYLCHOLINE-RECEPTOR FOR ORGANIC AND ALKALI-METAL CATIONS

We measured the permeability ratios (P(X)/P(Na)) of 3 wild-type, 1 hybrid, 2 subunit-deficient, and 22 mutant nicotinic receptors expressed in Xenopus oocytes for alkali metal and organic cations using shifts in the biionic reversal potential of the macroscopic current. Mutations at three positions (2', 6', 10') in M2 affected ion selectivity. Mutations at position 2' (alpha-Thr244, beta-Gly255, gamma-Thr253, delta-Ser258) near the intracellular end of M2 changed the organic cation permeability ratios as much as twofold and reduced P(Cs)/P(Na) and P(K)/P(Na) by 16-18%. Mutations at positions 6' and 10' increased the glycine ethyl ester/Na+ and glycine methyl ester/Na+ permeability ratios. Two subunit alterations also affected selectivity: omission of the delta-subunit reduced P(Ca)/P(Na) by 16%, and substitution of Xenopus delta for mouse delta increased P(guanidinium)/P(Na) more than twofold and reduced P(Ca)/P(Na) by 34% and P(Li)/P(Na) by 20%. The wild-type mouse receptor displayed a surprising interaction with the primary ammonium cations; relative permeability peaked at a chain length equal to four carbons. Analysis of the organic permeability ratios for the wild-type mouse, receptor shows that (a) the diameter of the narrowest part of the pore is 8.4 angstrom (b) the mouse receptor departs significantly from size selectivity for monovalent organic cations; and (c) lowering the temperature reduces P(guanidinium)/P(Na) by 38% and P(butylammonium)/P(Na) more than twofold. The results reinforce present views that positions -1' and 2' are the narrowest part of the pore and suggest that positions 6' and 10' align some permeant organic cations in the pore in an interaction similar to that with channel blocker, QX-222.