State Dependent Anionic Pore Currents Conducted by Single Countercharge Mutants in a Voltage-Sensing Phosphatase

Mutating gating charge residues in the S4 segment of voltage-sensing domains (VSDs) can cause ionic leak currents through the VSDs. These leak currents, known as gating pore or omega currents, play important pathophysiological role in many diseases. Here, we show that mutations in a key countercharge residue, D129, in the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) facilitate conduction of unique anionic omega currents. Neutralization of D129 causes a dramatic positive shift of activation, facilitates the formation of a continuous water path through the intermediate state VSD, and creates a positive electrostatic potential landscape inside the VSD leading to anion selectivity. Increasing the population or duration of the conducting state by a high external pH or an engineered Cd ²⁺ bridge markedly increases the current magnitude. Our findings uncover a new role of countercharge residues and could inform on the mechanisms of channelopathies linked to countercharge residue mutations.