Figure 15.

Effect of mutations at position 9′ of M2 on deactivation of the muscle nAChR and GLIC. (A and B) Macroscopic current responses to 1-ms pulses of 100-µM ACh recorded at –80 mV from the wild-type mouse muscle adult-type nAChR (A) and the αL9′A mutant (B) in the outside-out configuration. The solutions flowing through the two barrels of the perfusion tubing were (in mM) 142 KCl, 5.4 NaCl, 1.8 CaCl₂, 1.7 MgCl₂, and 10 HEPES/KOH, pH 7.4, with or without ACh. (C and D) Macroscopic current responses to 50-ms pulses of pH-4.5 solution recorded at –80 mV from wild-type GLIC (C) and the I9′A mutant (D) in the outside-out configuration. The solutions flowing through the two barrels of the perfusion tubing were (in mM) 142 KCl, 5.4 NaCl, 1.8 CaCl₂, 1.7 MgCl₂, and the pH was buffered with 10 HEPES/KOH (pH 7.4), 10 TABS/KOH (pH 9.0) or 10 acetic-acid/KOH (pH 4.5). In the case of the gain-of-function I9′A mutant, the (proton-gated) activity of the channel at pH 7.4 was relatively high, and hence, the pH of the solution applied during the “no-agonist” intervals was increased to 9.0. For all panels, the insets emphasize the time courses of deactivation; lines are fits to monoexponential-decay functions. (E) Mutant-construct deactivation time constants obtained from monoexponential fits normalized to the corresponding wild-type time constants. The plotted values are means obtained from 4 (L9′A nAChR) and 2 (I9′A GLIC) patches. An error bar (standard error) is only displayed for the nAChR; for GLIC, the two averaged values were 155 ms and 169 ms. All current traces shown in this figure are the averages of 10–25 consecutive responses recorded from representative patches.