Single channel currents were recorded from HEK 293 cells expressing recombinant mouse adult (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 estimated from open and closed time 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 AChRs slowing 400-fold. the rate constant of channel closing increased 3-fold, resulting in a decrease of the gating equilibrium constant of up to 1200-fold. In contrast to channel gating steps, ACh-binding steps are only modestly effected by alpha D200N. 3. Introduction of a potential glycosylation site in alpha D200N cannot account for the effect on channel gating because eliminating the consensus for glycosylation with 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 agonists carbamylcholine and tetramethylammonium also activate the alpha D200N AChR, but with channel opening rates even slower than with ACh. The agonist dependence of the opening rate constant is similar in alpha D200N and wild type AChRs. 5. AChRs containing D200N at just one of the two alpha-subunits show either small or large changes in the gating equilibrium constant, presumably due to the presence of the mutation at either the alpha delta or alpha epsilon/alpha gamma sites. The changes in free energy of channel gating show that the contribution of each binding site is nearly independent. However, the sites do not contribute equally to gating, as an alpha D200N mutation at the alpha epsilon or alpha gamma binding site slows channel opening relatively more than at the alpha delta site.
