Graded Tuning of Phosphatase Activity of VSP Coupled with the Intermediate State of the Voltage Sensor

Abstract

Voltage-sensing phosphatase (VSP) consists of the voltage sensor domain and the cytoplasmic phosphatase region. We have shown that the movement of the voltage sensor domain (VSD) is coupled to the phosphatase activity over a wide range of membrane potential (Sakata et al., 2011). However, it still remains elusive how the phosphatase activity is regulated by the movement VSD at the level of single molecule. One possible idea is that constant level of phosphatase activities is attained when VSD is in a fully activated state, and the number of molecules in the active state increases as the membrane potential becomes more positive. The other probability is that the enzymatic activity of single VSP proteins can be graded upon distinct activated states of VSD. In this case, partial activation of VSD leads to certain level of the phosphatase activity. To distinguish between two possibilities, we studied Dr-VSP with mutations in VSD, Dr-VSP(T156R/I165R), whose Q-V plot was able to be fitted by the sum of two Boltzmann equations. Analyses of ‘gating’ current and voltage clamp fluorometry showed that VSD of this mutant moves in two steps. The enzyme-defective mutant or the protein with mutation in the linker between VSD and the phosphatase domain also showed two-step movements of the VSD. Measurements of the phosphatase activity of Dr-VSP(T156R/I165R) revealed that both transitions of VSD activation increase the phosphatase activity. These suggest that the full activation of the voltage sensor is not necessary to exhibit the phosphatase activity, and the phosphatase activity at the single protein level could be graded dependent upon the magnitude of the movement of the VSD.