Offsetting voltage-dependent Kv1.5 channel opening through charged residue substitutions on top of the first transmembrane segment

Background: Voltage-dependent K+ (Kv) channels exist as tetramers of subunits consisting of six transmembrane segments (S1-S6). The S1 through S4 segments assemble into a voltage-sensing domain (VSD) that detects the membrane electric field. Therefore, the S4 is rich in positively charged amino acid residues forming the main component of the VSD. Upon membrane de- or repolarization, these positive charges, termed gating charges, cross the electric field, causing the VSD to change conformation. The S1-S3 segments surround the S4 and shape the electric field sensed by these gating charges.Materials and Methods: In the Shaker-type hKv1.5 channel residues C268 to R279 at the extracellular end of S1 and beginning of the S1-S2 linker were substituted by either a positive (lysine), a negative (glutamic acid), or a neutral (glutamine) residue. The charge substitution or charge introduction mutants were functionally analyzed by expressing the channels in Ltk- cells and performing patch-clamp current recordings.Results: At positions C268, L269, T271, and P273, a charge introduction was not tolerated, yielding no current expression. On the contrary, mutants D277K and R279E yielded channels with gating properties similar to wild type (WT) hKv1.5. The mutants E270Q, L272E, L272K, and F275K shifted the voltage dependence of channel activation toward more positive potentials compared to hKv1.5. However, this shift was accompanied by a shallower slope factor and altered activation and/or deactivation kinetics. Interestingly, at positions E274, R276, and E278, a charge substitution shifted the voltage dependence of channel opening without affecting channel kinetics, that is, exerting a surface charge effect.Conclusion: The cluster of charged residues at the top of S1 and the beginning of the S1-S2 linker are sufficiently close to the S4 and offset (polarize) the electric field sensed by the S4, contributing to the voltage dependence of channel activation.

Publication year:2022

Keywords:A1 Journal article

Accessibility:Closed