title: Coupling of the phosphatase activity of Ci-VSP to its voltage sensor activity over the entire range of voltage sensitivity.
abstract: The voltage sensing phosphatase Ci-VSP is composed of a voltage sensor domain (VSD) and a cytoplasmic phosphatase domain. Upon membrane depolarization, movement of the VSD triggers the enzyme's phosphatase activity. To gain further insight into its operating mechanism, we studied the PI(4,5)P2 phosphatase activity of Ci-VSP expressed in Xenopus oocytes over the entire range of VSD motion by assessing the activity of coexpressed Kir2.1 channels or the fluorescence signal from a pleckstrin homology domain fused with green fluorescent protein (GFP) (PHPLC-GFP). Both assays showed greater phosphatase activity at 125 mV than at 75 mV, which corresponds to 'sensing' charges that were 90% and 75% of maximum, respectively. On the other hand, the activity at 160 mV (corresponding to 98% of the maximum 'sensing' charge) was indistinguishable from that at 125 mV. Modelling the kinetics of the PHPLC-GFP fluorescence revealed that its time course was dependent on both the level of Ci-VSP expression and the diffusion of PHPLC-GFP beneath the plasma membrane. Enzyme activity was calculated by fitting the time course of PHPLC-GFP fluorescence into the model. The voltage dependence of the enzyme activity was superimposable on the Q-V curve, which is consistent with the idea that the enzyme activity is tightly coupled to VSD movement over the entire range of membrane potentials that elicit VSD movement.
authors.affiliation: Laboratory of Integrative Physiology, Department of Physiology, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan.
authors.forename: Souhei
authors.initials: S
authors.lastname: Sakata
authors.forename: Md Israil
authors.initials: MI
authors.lastname: Hossain
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Okamura
mesh_terms: D000818:Animals; D055162:Biocatalysis; D002938:Ciona intestinalis / Q000235:genetics; D055724:Electrophysiological Phenomena / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D015640:Ion Channel Gating / Q000502:physiology; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology*; D008954:Models, Biological; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D054803:Phospholipase C delta / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D018075:RNA, Complementary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: C489461:Kir2.1 channel; D019269:Phosphatidylinositol 4,5-Diphosphate; D024661:Potassium Channels, Inwardly Rectifying; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C509671:voltage-sensor-containing phosphatase, Ciona intestinalis; D010744:Phosphoric Monoester Hydrolases; D054803:Phospholipase C delta