Voltage Dependent Phosphatase Activity is Enhanced by Intracellular Acidification

Abstract

Voltage sensitive phosphatases (VSPs) are PI(4,5)P2/PI(3,4,5)P3-5- and PI(3,4)P2/PI(3,4,5)P3-3-phosphatases. For non-mammalian VSPs, this activity is regulated by membrane voltage via a voltage sensor domain (VSD). For mammalian VSPs the VSD seems insensitive to voltage changes yet still essential for control of the phosphatase activity. Under physiological conditions, the non-mammalian VSPs strongly deplete PI(4,5)P2 in a voltage dependent manner. The physiological regulator of mammalian VSP activity remains elusive. VSPs are suggested to play a role in fertilization and development, where changes in intracellular pH are known to occur. They are found to be expressed in tissues like kidney, stomach, sperm, and ovary which are known to undergo such pH changes. Therefore, we speculated that intracellular pH might modulate VSP activity.To test this hypothesis we performed whole-cell patch-clamp experiments in CHO cells expressing diverse VSPs and fluorescent PI(4,5)P2 reported domains, the latter allowing for monitoring VSP activity by means of total internal reflection microscopy (TIRF-M). The whole-cell patch-clamp configuration allowed for control not only over membrane voltage but also intracellular pH by dialysing the cell with solutions with the desired pH.We find that acidification of the cytoplasm results in increased PI(4,5)P2 depletion, accompanied by a shift of the apparent voltage dependence towards more negative potentials. An increase in intracellular pH has the opposite effect. The voltage dependence of sensing currents was unaffected by the pH changes, suggesting that alterations of the VSD are not causal for the observed changes in voltage dependent activity. Similar effects were observed in all tested VSPs We conclude that the overall activity of the phosphatase is enhanced under acidic and diminished under alkaline conditions. Kinetic modeling predicts a shift in apparent voltage dependence under these circumstances that is in agreement with the observed shift.In conclusion, we suggest that intracellular pH can play a role in the regulation of the activity of VSPs.This work was supported by a research grant of the University Medical Center Giessen and Marburg (UKGM32/2011MR) to C.R.H and by Deutsche Forschungsgemeinschaft (SFB593 TP A12) to D.O.