Creation of a reactive oxygen species-insensitive Kcv channel

The current of the minimal viral K+ channel Kcy(PCBV-1), heterologously expressed in Xenopus oocytes is strongly inhibited by reactive oxygen species (ROS) like H2O2. Possible targets for the ROS effect are two cysteines (C53 and C79) and four methionines (M1, M15, M23, and M26). The CS3A/C79A and M23L/M26L double mutations maintained the same ROS sensitivity as the wild type. However, M15L as a single mutant or in combination with C53A/C79A and/or M23L/M26L caused a complete loss of sensitivity to H2O2. These results indicate a prominent role of M15 at the cytosolic end of the outer transmembrane helix for gating of Kcvpav.i. Furthermore, even though the channel lacks a canonical voltage sensor, it exhibits a weak voltage sensitivity, resulting in a slight activation in the millisecond range after a voltage step to negative potentials. The M15L mutation inverts this kinetics into an inactivation, underlining the critical role of this residue for gating. The negative slope of the I-V curves of M15L is the same as in the wild type, indicating that the selectivity filter is not involved.