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J. Biol. Chem., Vol. 283, Issue 26, 18248-18259, June 27, 2008

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Enzyme Domain Affects the Movement of the Voltage Sensor in Ascidian and Zebrafish Voltage-sensing Phosphatases^*

Md. Israil Hossain^¶||1, Hirohide Iwasaki^¶||2, Yoshifumi Okochi^¶**, Mohamed Chahine, Shinichi Higashijima^¶||, Kuniaki Nagayama^¶||, and Yasushi Okamura^¶||**3

From the Department of Developmental Neurophysiology and Department of Nanoanatomy, Okazaki Institute for Integrative Bioscience, Higashiyama 5-1, Myodaiji-cho, Okazaki 444-8787, Japan, ^¶National Institute for Physiological Sciences, National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji-cho, Okazaki 444-8787, Japan, ^||Graduate Universities for Advanced Studies, Myodaiji-cho, Okazaki 444-8787, Japan, ^**Graduate School of Medicine, Osaka University, Yamada-oka 2-2, Suita 565-0871, Japan, and the Department of Medicine, Laval University and Le Centre de Recherche Université Laval Robert-Giffard, Québec G1J 2G3, Canada

The ascidian voltage-sensing phosphatase (Ci-VSP) consists of the voltage sensor domain (VSD) and a cytoplasmic phosphatase region that has significant homology to the phosphatase and tensin homolog deleted on chromosome TEN (PTEN).The phosphatase activity of Ci-VSP is modified by the conformational change of the VSD. In many proteins, two protein modules are bidirectionally coupled, but it is unknown whether the phosphatase domain could affect the movement of the VSD in VSP. We addressed this issue by whole-cell patch recording of gating currents from a teleost VSP (Dr-VSP) cloned from Danio rerio expressed in tsA201 cells. Replacement of a critical cysteine residue, in the phosphatase active center of Dr-VSP, by serine sharpened both ON- and OFF-gating currents. Similar changes were produced by treatment with phosphatase inhibitors, pervanadate and orthovanadate, that constitutively bind to cysteine in the active catalytic center of phosphatases. The distinct kinetics of gating currents dependent on enzyme activity were not because of altered phosphatidylinositol 4,5-bisphosphate levels, because the kinetics of gating current did not change by depletion of phosphatidylinositol 4,5-bisphosphate, as reported by coexpressed KCNQ2/3 channels. These results indicate that the movement of the VSD is influenced by the enzymatic state of the cytoplasmic domain, providing an important clue for understanding mechanisms of coupling between the VSD and its effector.

Received for publication, July 27, 2007 , and in revised form, March 17, 2008.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB308476.

^* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to Y. O. and H. I.). Some data relevant to this paper were included in abstract form at the Biophysical Society meeting in 2007 (15). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5.

¹ Supported by honors scholarship for self-funded international students from JASSO.

² Present address: Dept. of Molecular and Cellular Biology, Harvard University, 7 Divinity Ave., Fairchild 132, Cambridge, MA 02138.

³ To whom correspondence should be addressed: Dept. of Developmental Neurophysiology, Okazaki Institute for Integrative Bioscience, National Institute for Physiological Sciences, Higashiyama 5-1, Okazaki, 444-8787, Japan. Tel.: 81-564-59-5256; Fax: 81-564-59-5259; E-mail: yokamura{at}phys2.med.osaka-u.ac.jp.

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				H. Iwasaki, Y. Murata, Y. Kim, Md. I. Hossain, C. A. Worby, J. E. Dixon, T. McCormack, T. Sasaki, and Y. Okamura A voltage-sensing phosphatase, Ci-VSP, which shares sequence identity with PTEN, dephosphorylates phosphatidylinositol 4,5-bisphosphate PNAS, June 10, 2008; 105(23): 7970 - 7975. [Abstract] [Full Text] [PDF]