抄録
Voltage sensor domain (VSD) regulates ion permeation and enzyme activity. It remains unknown how coupling mechanisms are similar between voltage-gated channels and voltage-sensing phosphatases, VSPs. The voltage dependency of the movement of the voltage sensor of Ci-VSP is less steep than that of voltage-gated channels, and is not saturated even over 100 mV. To clarify the voltage range at which enzyme activity is saturated, kinetics of PIP2 depletion was compared with confocal microcope at distinct membrane potentials by monitoring cell surface translocation of GFP fused with a PH-domain (PHD-GFP). The change of translocation of PHD-GFP occurred at time constant of 22.8 +- 15.0 sec (n=5) at 50 mV and 7.7 +- 2.8 sec (n=5) at 100 mV, whereas no significant difference was found between 100 mV and 150 mV, possibly due to slow diffusion speed of PHD-GFP protein. As alternative approach, kinetics of outward current through engineered inward-rectifier potassium channel, Kir2.1(4mut), with mutations at sites critical for inward-rectification (Fujiwara & Kubo, J. Physiol., 2002) was electrophysiologically measured. Decay of outward current through Kir2.1(4mut) became sharper as the voltage increases from 50 mV to 100 mV, and also from 100 mV to 170 mV. However, decay kinetics was similar at higher range, suggesting that enzyme activity is saturated. This provides potent evidence that voltage-dependency of downstream effecter activity is shifted rightward with reference to that of the VSD as in voltage-gated ion channels. [J Physiol Sci. 2008;58 Suppl:S77]
