Addition Of A Charged Residue At Position 363 Focuses The Electric Field Sensed By S4 During Activation Of Shaker K-Channels

Voltage-gated potassium channels contain a voltage sensor domain in each of its four subunits that confers exquisite sensibility to the trasmembrane electric field. The fourth transmembrane segment (S4) of a Shaker K-channel subunit has seven highly conserved basic amino acids, periodically spaced every two hydrophobic residues. The four outermost arginine residues (R1-R4) in S4 move through most of the electric field upon depolarization. This charge movement (12-13 e0 per channel) is tightly coupled to the conformational change leading the opening of the potassium conduction pathway. Any residue located within the R1-R4 limits would be dragged through the electric field, and if charged would contribute to the charge movement. Surprisingly, when we replaced V363 (located between R1 and R2) by either an arginine or an aspartate, both charge adding mutations decreased the effective valence of opening by 50%. This reduction in effective valence was not attributable to an uncoupling between charge movement and channel opening because the Q-V curve remained preceding the G-V curve. To test if the added charges promote a remodeling of the electric field sensed by S4, we assessed the state-dependency of external accessibility of R1 or R2 in the presence of the additional charge. Then we tested the accessibility to methanethiosulfonate derivates in R1C/V363R, R1C/V363D, R2C/V363R or R2C/V363D double-mutants. We measured the rates of cysteine modification within a ten-fold change in the overall open probability for each mutant. Unlike the R1C control, the modification rates were not state-dependent for R1C/V363X double-mutants, while they remained state-dependent for R2C/V363X double-mutants as the R2C control. These results suggest that in channels carrying a charged residue at position 363, R362 remains outside the field during voltage dependent activation. Funded by ACT-46.