2024-03-28T10:58:06Zhttps://eprints.lib.hokudai.ac.jp/dspace-oai/requestoai:eprints.lib.hokudai.ac.jp:2115/820322022-11-17T02:08:08Zhdl_2115_54838hdl_2115_76083hdl_2115_54822hdl_2115_54823hdl_2115_20124Analysis of the Substrate Uptake and Release Reactions of the Light-Driven Sodium-Pump Rhodopsin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・放出反応の解析村部, 圭祐Murabe, Keisuke出村, 誠黒川, 孝幸菊川, 峰志open access400For 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.(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)Hokkaido University2021-03-25engdoctoral thesisVoRhttps://doi.org/10.14943/doctoral.k14406http://hdl.handle.net/2115/8203210.14943/doctoral.k14406http://hdl.handle.net/2115/8203045p甲第14406号博士(ソフトマター科学)2021-03-2510101北海道大学https://eprints.lib.hokudai.ac.jp/dspace/bitstream/2115/82032/1/Keisuke_Murabe.pdfapplication/pdf2.04 MB2021-03-25