ja0c07264_si_001.pdf (1.15 MB)
Direct Detection of the Substrate Uptake and Release Reactions of the Light-Driven Sodium-Pump Rhodopsin
journal contribution
posted on 2020-09-07, 06:14 authored by Keisuke Murabe, Takashi Tsukamoto, Tomoyasu Aizawa, Makoto Demura, Takashi KikukawaFor
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. We 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 employed to detect Na+ uptake and release.
When one side of the membrane was covered by the lipid-reconstituted
NaR, continuous illumination induced an increase in membrane potential,
which reflected Na+ uptake by the photolyzed NaR. Via use
of nanosecond laser pulses, two kinds of data were obtained during
a single transport cycle: one was the flash-induced absorbance change
in NaR 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.
Their comparison clearly indicated that Na+ is captured
and released during the formation and decay of the O intermediate,
the red-shifted intermediate that appears in the latter half of the
transport cycle.