A Voltage Sensitive Phosphatase from Xenopus Laevis Testis

Abstract

Voltage sensitive phosphatases (VSPs) are transmembrane proteins comprised of a voltage sensor domain characteristic of an ion channel, coupled to a phosphatidylinositol phosphatase; their phosphatase activity is activated by membrane depolarization (Murata and Okamura, 2007, J. Physiol. 583:875-889). Because VSPs are expressed predominantly in sperm, it has been proposed that they might function in the voltage-dependent regulation of sperm-egg membrane fusion, which in many species provides a fast block to polyspermy. To characterize the properties of a VSP from a species amenable to transgenesis and fertilization studies, we identified a VSP homolog from Xenopus laevis testis and expressed it in Xenopus oocytes, together with the fluorescent PIP2 sensor PLCδPH-GFP. Using a photodiode to measure PLCδPH-GFP fluorescence from the pigmented surface of voltage clamped oocytes, we showed that XlVSP enzymatic activity is regulated over a range of membrane potentials (50% activation at ∼ +10 mV) similar to those that regulate sperm-egg fusion. In agreement with previous studies of ascidian and zebrafish VSPs (Hossain et al., 2008, J. Biol. Chem. 283: 18248-59), an R152Q point mutation shifted the XlVSP activation curve towards more hyperpolarized membrane potentials (50% activation at ∼ −12mV). Future studies of the voltage dependence of fertilization using sperm from transgenic frogs expressing XlVSPR152Q should allow investigation of the functional significance of voltage sensitive phosphatases in sperm-egg fusion.