Stabilization of the Relaxed State of the Voltage Sensing Domain of Shaker

Abstract

Segments S4 and S5 in Voltage Gated Channels potassium channels are contiguous and specific residues of these segments get in atomic proximity in a state-dependent way (Laineet al., 2003; Lewis et al., 2008). In Shaker, the double mutation R362H+A419H stabilizes the conducting state of the channels when a metal bridge is formed in the presence of Zn2+ (Laineet al., 2003). These results were obtained from ionic conduction experiments but gave no direct information on the dynamics of the Voltage Sensing Domain (VSD) of Shaker. As a proxy for the movement of the VSD, we studied the proton currents through the VSD that results by the double mutation R362H+A419H, on the ultra-fast-inactivating Shaker W434F. When the holding potential (HP) was 0 mV, the current-voltage relation of the proton current (Ip-V) was shifted towards negative potentials as compared to the Ip-V when HP was −90 mV, as expected from the relaxation that the VSD undergoes at maintained depolarization. When HP was 0 mV, the proton current was decreased and the Ip-V was further shifted by increasing the concentration of Ni2+ or Zn2+ (10 μM-100 μM). In contrast, no changes were observed in the Ip-V voltage dependence with Ni2+ or Zn2+ when holding at −90 mV. In the presence of Ni2+ or Zn2+ the proton current showed a second slower kinetic component, whose relative amplitude was increased with an increase in Ni2+or Zn2. Fluorescence recordings with a probe in M356C showed that Zn2+ decreased the rate of TMRM dequenching when pulsing to negative potentials from an HP of 0 mV, consistent with the proton current results. These observations indicate that the metal bridge between R362H and A419H stabilizes the relaxed state of the VSD (Support NIHGM030376).