Contribution of the central hydrophobic residue in the PXP motif of voltage-dependent K⁺ channels to S6 flexibility and gating properties

Shaker-like (K_V1.1) channels contain a highly conserved Pro-Val-Pro (PVP) motif at the base of S6 that produces a kink in the S6 helices and provides a flexible element thought to be essential for channel gating. The role of proline-induced kinks in transmembrane helices is well known, but the contribution of the small hydrophobic valine between these two prolines is not known, and interestingly, Shab-like (K_V2.1) channels possess an isoleucine at this position (PIP). Here we show that the exact nature of this central hydrophobic residue within the PXP motif confers unique functional properties to K_V1 channels, including changes in activation and deactivation kinetics, voltage-dependent properties and open probabilities, but single-channel conductance and cell expression levels are not affected. In support of these functional changes, molecular dynamic simulations demonstrate that valine and isoleucine contribute differently to S6 flexibility within this motif. These results therefore indicate that the nature of the central hydrophobic residue in the PXP motif is an important functional determinant of K_V channel gating by contributing, at least in part, to the relative flexibility of this motif.