Interaction Between Two Domains in the AChR Gating Reaction

Abstract

The AChR is a large ion channel that isomerizes between non-conducting and conducting conformations. Residue αA96 is located in loop 5 (loop A) near the agonist-binding site, which moves at the outset of the channel-opening process (the Φ value for the adjacent residue αD97 is 0.93). Side chain substitutions at nearby (<0.45 nm, 2QC1.pdb) residue αY127 (β-strand 6) change the gating equilibrium constant (Keq) by up to 290,000-fold (Φ=0.77). This suggests that αY127 moves in concert with the lower part of the extracellular domain, after the motion of loop 5. αD97 and αY127 are not coupled energetically. We tested the hypothesis that αA96 and αY127 energetically link the first two Φ-blocks, to propagate the opening conformational wave from the upper to the lower part of the extracellular domain. We mutated residue αA96 (C, F, K, L, N, Q) and measured single-channel gating kinetics (mouse α2βδe, cell-attached, -100 mV, 20 mM choline, PBS, 23°C). The Φ-value for αA96 is 0.90, indicating that it moves at the onset of channel gating along with other residues in loop 5. αA96N showed the largest change in Keq (∼900-fold) and markedly increased unliganded gating. Next, we performed mutant cycle analysis to test for energetic coupling between αA96 and αY127. Keq for the double mutant αA96K+αY127E is 18-fold greater than the wt, where the effects of the single mutants, if additive, predict one that is 9.2-fold smaller. This corresponds to a coupling free energy of -3.1 kcal/mol. Similarly, Keq for the double mutant αA96C+αY127C is 213-fold greater than the wt, whereas a value 1.7-fold smaller is predicted assuming independence (coupling free energy of -3.6 kcal/mol). These are large interaction energies that suggest αA96 and αY127 form a key energetic link between the first and second Φ-blocks.