Engineered Voltage Sensing Phosphatases: What do they tell us about the Gating Mechanism?

Abstract

Voltage sensing phosphatases (VSPs) contain a phosphoinositide phosphatase domain (PD), which is under the control of a voltage sensor domain (VSD). Here, the underlying coupling mechanism is not fully understood. Recently, Liu et al. (NSMB 2012) and Hobiger et al. (Biophys J 2012, PLoS one 2013) proposed for Ciona intestinalis (Ci-) VSP an interaction of positively charged amino acid residues in the linker, which connects the PD to the VSD, with the negatively charged residue Asp400 in the ‘TI’ loop of the PD. In light of these findings we revisited the engineered VSPs PTENCiV (Lacroix et al., JBC 2011) and hVSP1CiV (Halaszovich et al., J Lipid Res 2012), chimeras consisting of Ci-VSP's VSD and the PD of PTEN or hVSP1, respectively. In those chimeras Asp400 (Ci-VSP numbering) is not conserved. Nonetheless, they show robust voltage dependent phosphatase activity. Mutations within the linker showed effects mirroring those previously reported for Ci-VSP, suggesting a gating mechanism similar to the one found in wild-type Ci-VSP. We mutated Asp400 in Ci-VSP to Asn or Arg and the corresponding Asn (hVSP1) or Arg (PTEN) in the chimeric enzymes to Asp and measured their voltage dependent enzymatic activity as well as sensing currents to elucidate the importance of Asp400 for VSD-PD-coupling. We propose that the interactions involving Asp400 described by Liu et al. and Hobiger et al. are not a necessary prerequisite for the voltage dependent activation of VSPs, but allow for a more efficient VSD-to-PD coupling. Further work will be required to establish the essential interactions between VSP, linker, and PD. Supported by Deutsche Forschungsgemeinschaft (DFG, grant SFB 593, TP12 to D.O.) and University Medical Center Giessen and Marburg (UKGM, grant 32/2011MR to C.R.H.)