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. 2011 Apr 20;100(8):1930–1939. doi: 10.1016/j.bpj.2011.02.046

Figure 7.

Figure 7

Disruption of the Ile (αI93) and His (αH94) residues immediately upstream of the critical PHA-1 Gly (αG95) also leads to voltage dependence reminiscent of mutation of the conserved Trp (αW112) residue at the intracellular base of TM1. (A) Families of macroscopic Na+ currents from CHO cells expressing mENaC containing α-subunits with T92C (left), I93C (left middle), H94C (right middle), or A96C (right) substitution. The relative position to the critical Gly (G95) is indicated. Currents evoked by progressive 20 mV voltage steps from a holding potential of 0 mV up to 100 mV and down to −200 mV. For these experiments, the bath and pipette [Na+] were symmetrical at 150 mM. (B) Macroscopic I/V relations for CHO cells expressing wild-type (black lines) and mutant (gray lines) mENaC containing α-subunits with T92C (left), I93C (left middle), H94C (right middle), or A96C (right) substitution. The I/V relations were generated from experiments similar to that in panel A. For presentation, current was normalized to current at −100 mV. (C) Summary graph of (amiloride-sensitive) current density at steady state at −80 mV for voltage-clamped CHO cells (as in Fig. 6A) expressing wild-type mENaC and mENaC containing α-subunits with T92C, I93C, H94C, G95C, or A96C substitution. ∗ Significant (P < 0.05) decrease versus wild-type mENaC. (D) Steady-state G-V relations were fitted with the Boltzmann equation for CHO cells expressing mENaC containing α-subunits with I93C (gray triangle), H94C (open circle), G95C (black box), A96C (open box), or W112C (dashed line) substitution. Data are from experiments identical to that shown in panel A. (E) Typical activation kinetics at −200 mV for normalized (to maximums at steady state) macroscopic Na+ currents carried by mENaC containing α-subunits with I93C, H94C, or G95C substitution. Current for I93C is shown at both 1 s (black line) and 10 s (gray line; noted with arrow) timescales. Inset shows mean τactivation at −60 mV for the respective mutants. For B–D and inset in E, n ≥ 6 for each group.