Effect of a sensing charge mutation on the deactivation of KV7.2 channels

Potassium-selective, voltage-gated channels of the KV7 family are critical regulators of electrical excitability in many cell types. Removing the outermost putative sensing charge (R198) of the human KV7.2 shifts its activation voltage dependence toward more negative potentials. This suggests that removing a charge "at the top" of the fourth (S4) segment of the voltage-sensing domain facilitates activation. Here, we hypothesized that restoring that charge would bring back the activation to its normal voltage range. We introduced the mutation R198H in KV7.2 with the idea that titrating the introduced histidine with protons would reinstate the sensing charge. As predicted, the mutant's activation voltage dependence changed as a function of the external pH (pHEXT) while modest changes in the activation voltage dependence were observed with the wild-type (WT) channel. On the other hand, the deactivation kinetics of the R198H mutant was remarkably sensitive to pHEXT changes, readily deactivating at pHEXT 6, while becoming slower to deactivate at pHEXT 8. In contrast, the KV7.2 WT displayed modest changes in the deactivation kinetics as a function of pHEXT. This suggested that the charge of residue 198 was critical for deactivation. However, in a surprising turn, the mutant R198Q-a non-titratable mutation-also displayed a high pHEXT sensitivity activity. We thus concluded that rather than the charge at position 198, the protonation status of the channel's extracellular face modulates the open channel stabilization and that the charge of residue 198 is required for the voltage sensor to effectively deactivate the channel, overcoming the stabilizing effect of high pHEXT.