open access

https://doi.org/10.14943/doctoral.k14406

https://hdl.handle.net/2115/82032

光駆動ナトリウムポンプロドプシンによる基質の取込み・放出反応の解析

英語

2021-03-25

Hokkaido University

45p

For membrane transporters, substrate uptake and release reactions are major events during their transport cycles. Despite the functional importance of these events, it is difficult to identify their relevant structural intermediates because of the requirements of the experimental methods, which are to detect the timing of the formation and decay of intermediates and to detect the timing of substrate uptake and release. I report successfully achieving this for the light driven Na+ pump rhodopsin (NaR). Here, a Na+-selective membrane, which consists of polyvinyl chloride and a Na+ ionophore, was employe d to detect Na+ uptake and release. Firstly, I confirmed the Na+-concentration response of the bare Na+ selective membrane. This membrane showed good response over a wide Na+ concentration range. When one side of the membrane was covered by the lipid-reconstituted NaR, continuous illumination induced an increase in membrane potential, reflecting Na+ uptake by the photolyzed NaR. The s ame measurements with the NaR-disabled mutant and the H+ pump rhodopsin showed no change in membrane potential. These results negated the contribution of electrogenicity to the membrane potential due to the NaR orientation in a specific direction, and showed that this membrane only detected the change in Na+ concentration. By using nanosecond laser pulses, two kinds of data were obtained during a single transport cycle: one was the flash induced absorbance change in NaR in order to detect the formation and decay of structural intermediates, and the other was the flash induced change in membrane potential, which reflects the transient Na+ uptake and release reactions. The flash induced absorbance change showed the acceleration of the formation of O intermediate as the Na+ concentration increased . The flash-induced potential change showed positive change in membrane potential during a single transport cycle. Their comparison clearly indicated that Na is captured and released during the formation and decay of the O intermediate, the redshifted intermediate which appears in the latter half of the transport cycle.

2021-03-25

甲第14406号

博士(ソフトマター科学)

(主査) 教授 出村 誠, 教授 黒川 孝幸, 講師 菊川 峰志

生命科学院（ソフトマター専攻）

博士論文

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