State-Dependent Lipid Interactions Couple the Conformations of the Voltage-Sensing and Pore-Gate Domains

Abstract

Coupling between the voltage-sensing domain (VSD) and pore-gate domain (PGD) is required for the voltage-dependent gating of ion channels, but the molecular mechanisms of coupling are unclear. Previous studies have identified protein-protein interactions that are important for coupling, while structural and recent functional data demonstrate that membrane lipids also play a role. In a recent study of Kv7.1, we found that phosphatidylinositol 4,5-bisphosphate (PIP2) binding at the VSD-PD interface is required to couple the activated-state of the VSD to the open-state of the PD. We devised a method to directly measure PIP2 mediated coupling and an allosteric framework for describing such coupling. These advances provide new tools to investigate the mechanisms of VSD-PGD coupling. We also identified a putative PIP2 binding site and found that mutations of residues within this site reduce PIP2-mediated coupling. Paradoxically, a set of mutations near the PIP2 binding site increased the macroscopic current. Using a combined computational and experimental approach to study these gain of function mutations, we now identify a PIP2-interaction that is preferred by the resting-closed channel. Using the KCNE1 accessory subunit as an experimental tool, we are able to resolve the functional effects of this resting-closed state interaction. These results allow us to propose a novel mechanism for voltage-dependent gating in which repositioning of cofactor lipids at the VSD-PD represents a critical step in the transitions between resting-closed and activated-open states. This model can be used to explain the phenomena of cooperativity and concerted motion in voltage-gated channels.