Stabilization of the relaxed state of the voltage sensing domain of Shaker
Carlos A. Villalba-Galea: 0000-0002-6489-4651
Conference Title/Conference Publication
Biophysical Society 54th Annual Meeting
San Francisco, CA
February 20-24, 2010
Date of Presentation
3, Supplement 1
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 (Lainéet 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+ (Lainéet 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).
Villalba-Galea, Carlos A.; Frezza, Ludivine; and Bezanilla, Francisco, "Stabilization of the relaxed state of the voltage sensing domain of Shaker" (2010). School of Pharmacy Faculty Presentations. 433.