Effects of mutations to the lidocaine-binding site of ELIC. (A–C) Macroscopic current responses to 5-s pulses of 10 mM cysteamine recorded at –80 mV from 3 M2 α-helix mutants 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 cysteamine. The trace shown in C is the average of 27 responses recorded from 7 patches. (D and E) Peak-current amplitudes and time constants of desensitization normalized to wild-type values. The plotted values are means obtained from 3 (Y14′A), 4 (Y17′A), and 7 (Y14′A + Y17′A) patches; error bars are the corresponding standard errors. The desensitization time courses recorded from the Y14′A + Y17′A double mutant had low peak-current amplitudes, and hence, a total of 27 traces from 7 patches were averaged and fitted (Table 3); a proper fit required two (rather than one) exponential-decay components. (F–H) Single-channel inward currents elicited by 10 mM cysteamine recorded at –80 mV in the outside-out configuration; openings are downward deflections. The external solution was (in mM) 142 KCl, 5.4 NaCl, 1.8 CaCl2, 1.7 MgCl2, 10 cysteamine, and 10 HEPES/KOH, pH 7.4. For comparison, single-channel traces recorded from the wild-type channel under identical experimental conditions are shown in Fig. 6 B, top. (I and J) Macroscopic current responses to 5-s pulses of 10 mM cysteamine in the continuous presence of 5 mM lidocaine recorded at –80 mV from 2 mutants (in the M3 and M2 α-helices, respectively) in the outside-out configuration. The solutions flowing through the two barrels of the perfusion tubing were as in A–C, with the addition of 5 mM lidocaine. In the schematic representation of the theta-tubing perfusion, arrows indicate the application of lidocaine.
