Figure 7

Effects of extracellular acidosis on current amplitude abolished by double mutation of E575 and H578. (ai-ci) Membrane current traces of single point mutant (ai and bi) hERG1a channel currents and the double mutant E575Q/H578N, evoked by step depolarization to +20 mV and repolarization to −40 mV (holding potential −80 mV). Application of pH 6.3 to hERG1a-E575Q had little effect on macroscopic current amplitude positive to +20 mV, but significantly accelerated current deactivation. Greater inhibition of macroscopic current by pH 6.3 of current mediated by hERG1a-H578N was observed, with less effect on the time-course of current deactivation. In contrast, hERG1a-E575Q/H578N-mediated current was augmented by extracellular acidosis at +20 mV, with a clear acceleration of current deactivation. (aii-bii) Current-voltage relationships for the two single point mutants and the double mutant of hERG1a channels. Extracellular acidosis reduced the amplitude of evoked current at potentials negative to +20 mV, with little effect at more depolarized levels. In contrast, current amplitude mediated by hERG1a-E575Q/H578N was unaffected at potentials negative to +20 mV, but was augmented at more depolarized levels. The normalized current-voltage relationship for wildtype hERG1a current is shown on each plot as superimposed dashed lines (black, pH 7.4; grey, pH 6.3). (aiii-ciii) Activation curves showing that each single and double mutant hERG1a channel current was affected by application of pH 6.3 extracellular solution, with voltage dependence of activation being shifted positive by acidosis. (d–f) Bar charts showing the effect of extracellular acidosis on WT, hERG1a-E575Q, hERG1a-H578N and hERG1a-E575Q/H578N on the amplitude of the end pulse current evoked by a step to +20 mV (VH −80 mV) (d), the fast time component (τ_Fast) (e), and slow time component (τ_Slow) (f), of current deactivation. Numbers in brackets indicate cell replicates.