Phe233 in the Voltage-Sensor is Rate Limiting for Channel Closure but not for the Opening

Abstract

During activation, the charged S4 segment in the voltage sensor domain (VSD) of voltage-gated ion channels is required to translate across a hydrophobic zone. This constitutes a thin aperture clearly separating the open/relaxed and closed/resting states. The nature of this barrier is critical for channel function, and has been the focus of much attention. Previously, we identified the single preserved residue F233 (F290 in Shaker) as a structural barrier for the gating charges, that uniquely determines intermediate state and substitutions modulate the deactivation barrier. A fundamental understanding of the S4 activation/deactivation barrier as well as kinetics is an important remaining challenge to decipher gating. Here, we study the free barrier and kinetics of the VSD through combining in-vitro and in-silico experiments. We used site-directed mutagenesis and measured the voltage-dependence in-vitro to study the effect of F290L compared to the wild-type (WT). In parallel, molecular dynamics simulations allowed us to identify residues interacting with the phenyl ring. Through in-silico mutations we show their impact on the barrier, as well as the structural and kinetics effects for the phenyl ring orientation during the first step of deactivation and the (reverse) last step of the activation. Strikingly, the channel closing transition shows a huge speedup from the F290L mutant (1ms, WT 10ms), in contrast to the opening that is completely unaffected. This indicates that F233 is only rate-limiting for the channel closure, but not opening and suggests different kinetics for the activation/deactivation barriers. Additionally the ring is clearly stabilized through vdW interactions with surrounding hydrophobic residues, and appears to always open by upward rotation both for activation/deactivation. This upward rotation suggests a model where the closing is possibly a mostly entropic process, while opening would be largely enthalpic.