Electron Spin Echo Envelope Modulation (ESEEM) Reveals the Footprint of the Voltage Sensor on the KvAP Pore Domain

Abstract

In voltage gated potassium channels, two interfaces between the central pore domain (PD) and the peripheral voltage sensor domain (VSD) must exist for the efficient transduction of membrane potential changes into mechanical opening of the gate. The first interface, located between the S4-S5 linker (VSD) and the S6 helix (PD), couples VSD motion to PD motion. Additionally, a strong secondary interface is mechanistically required to act as an anchor point between the domains so that force can be efficiently transduced to the PD. However, no such interface is apparent in any current crystal structure. As multiple studies have identified the S1 helix as the likely point of anchoring of the VSD, we set out to determine the interaction footprint of the VSD on the PD using ESEEM spectroscopy. We have previously demonstrated that deuterium ESEEM is well suited to investigate the interaction of membrane proteins with their surrounding environment. In the present study, we determine the water accessibility profile of the KvAP PD in the presence and absence of the VSD. We show that a region of the PD near the monomer interface demonstrates decreased deuterium coupling in the presence of the VSD compared to what would be expected based on residue immersion depth. Furthermore, the observed deuterium coupling at this region increases to expected levels upon removal of the VSD. We conclude that the protected region of the PD represents the interaction footprint of the VSD on the surface of the pore.