BINDING-SITES CONTRIBUTE UNEQUALLY TO THE GATING OF MOUSE NICOTINIC ALPHA-D200N ACETYLCHOLINE-RECEPTORS

1. Single channel currents were recorded from HEK 293 cells expressing recombinant mouse adult (alpha(2) beta delta epsilon) and embryonic ( alpha(2) beta delta gamma) acetylcholine receptors (AChRs) containing a mutation at residue D200 of the alpha-subunit. Rate and equilibrium constants for AChR activation were established from open and closed ti mes obtained over a range of ACh concentrations. 2. Mutation of alpha D200 to asparagine (alpha D200N) dramatically slows the rate constant of channel opening, with adult AChRs slowing 100-fold and embryonic AC hRs slowing 400-fold. The rate constant of channel closing increases 3 -fold, resulting in a decrease of the gating equilibrium constant of u p to 1200-fold. In contrast to channel gating steps, ACh-binding steps are only modestly effected by alpha D200N. 3. Introduction of a poten tial glycosylation site in alpha D200N cannot account for the effect o n channel gating because eliminating the consensus for glycosylation w ith the mutation alpha D200N + T202V fails to restore efficient gating . Gating is similarly impaired with the substitutions of E, K and Q at position alpha 200. 4. The agonist carbamylcholine and tetramethylamm onium also activate the alpha D200N AChR, but with channel opening rat es even slower than with ACh. The agonist dependence of the opening ra te constant is similar in alpha D200N and wild type AChRs. 5. AChRs co ntaining D200N at just one of the two alpha-subunits show either small or large changes in the gating equilibrium constant, presumably due t o the presence of the mutation at either the alpha delta or alpha epsi lon/alpha gamma sites. The changes in free energy of channel gating sh ow that the contribution of each binding site in nearly independent. H owever, the sites do not contribute equally to gating, as an alpha D20 0N mutation at the alpha epsilon or alpha gamma binding site slows cha nnel opening relatively more than at the alpha delta site.