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Heterologous Expression of Pharaonis Halorhodopsin in Xenopus laevis Oocytes and Electrophysiological Characterization of Its Light-Driven Cl- Pump Activity

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Title: Heterologous Expression of Pharaonis Halorhodopsin in Xenopus laevis Oocytes and Electrophysiological Characterization of Its Light-Driven Cl- Pump Activity
Authors: Seki, Akiteru Browse this author
Miyauchi, Seiji Browse this author
Hayashi, Saori Browse this author
Kikukawa, Takashi Browse this author →KAKEN DB
Kubo, Megumi Browse this author
Demura, Makoto Browse this author →KAKEN DB
Ganapathy, Vadivel Browse this author
Kamo, Naoki Browse this author
Issue Date: 1-Apr-2007
Publisher: The Biophysical Society
Journal Title: Biophysical Journal
Volume: 92
Issue: 7
Start Page: 2559
End Page: 2569
Publisher DOI: 10.1529/biophysj.106.093153
PMID: 17208978
Abstract: Natronomonas pharaonis halorhodopsin (pHR) is an archaeal rhodopsin functioning as an inward-directed, light-driven Cl– pump. To characterize the electrophysiological features of the Cl– pump activity of pHR, we expressed pHR in Xenopus laevis oocytes and analyzed its photoinduced Cl– pump activity using the two-electrode voltage-clamp technique. Photoinduced outward currents were observed only in the presence of Cl–, Br–, I–, , and SCN–, but not in control oocytes, indicating that photoinduced anion currents were mediated by pHR. The relationship between photoinduced Cl– current via pHR and the light intensity was linear, demonstrating that transport of Cl– is driven by a single-photon reaction and that the steady-state current is proportional to the excited pHR molecule. The current-voltage relationship for pHR-mediated photoinduced currents was also linear between –150 mV and +50 mV. The slope of the line describing the current-voltage relationship increased as the number of the excited pHR molecules was increased by the light intensity. The reversal potential (VR) for Cl– as the substrate for the anion pump activity of pHR was about –400 mV. The value for VR was independent of light intensity, meaning that the VR reflects the intrinsic value of the excited pHR molecule. The value of VR changed significantly for the R123K mutant of pHR. We also show that the Cl– pump activity of pHR can generate a substantial negative membrane potential, indicating that pHR is a very potent Cl– pump. We have also analyzed the kinetics of voltage-dependent Cl– pump activity as well as that of the photocycle. Based on these data, a kinetic model for voltage-dependent Cl– transport via pHR is presented.
Type: article (author version)
URI: http://hdl.handle.net/2115/22081
Appears in Collections:薬学研究院 (Faculty of Pharmaceutical Sciences) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 宮内 正二

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