title: Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid.
abstract: The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane.
authors.affiliation: Laboratory of Integrative Physiology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan; Institute for Academic Initiatives, Osaka University, Suita, Osaka 565-0871, Japan sakatas@osaka-med.ac.jp yokamura@phys2.med.osaka-u.ac.jp.
authors.forename: Souhei
authors.initials: S
authors.lastname: Sakata
authors.affiliation: Laboratory of Integrative Physiology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan;
authors.forename: Yuka
authors.initials: Y
authors.lastname: Jinno
authors.affiliation: Laboratory of Integrative Physiology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan;
authors.forename: Akira
authors.initials: A
authors.lastname: Kawanabe
authors.affiliation: Laboratory of Integrative Physiology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan; sakatas@osaka-med.ac.jp yokamura@phys2.med.osaka-u.ac.jp.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Okamura
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002938:Ciona intestinalis; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D014981:Xenopus; D015091:beta-Alanine / Q000031:analogs & derivatives*
chemical_list: C546053:3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid; D015091:beta-Alanine; C509671:voltage-sensor-containing phosphatase, Ciona intestinalis; D010744:Phosphoric Monoester Hydrolases
keywords: VSP; phosphatase; unnatural amino acid