title: The photochemistry of sodium ion pump rhodopsin observed by watermarked femto- to submillisecond stimulated Raman spectroscopy.
abstract: Krokinobacter rhodopsin 2 (KR2) is a recently discovered light-driven Na(+) pump that holds significant promise for application as a neural silencer in optogenetics. KR2 transports Na(+) (in NaCl solution) or H(+) (in larger cation solution, e.g. in CsCl) during its photocycle. Here, we investigate the photochemistry of KR2 with the recently developed watermarked, baseline-free femto- to submillisecond transient stimulated Raman spectroscopy (TSRS), which enables us to investigate retinal chromophore dynamics in real time with high spectral resolution over a large time range. We propose a new photocycle from femtoseconds to submilliseconds: J (formed in ∼200 fs) → K (∼3 ps) → K/L1 (∼20 ps) → K/L2 (∼30 ns) → L/M (∼20 μs). KR2 binds a Na(+) ion that is not transported on the extracellular side, of which the function is unclear. We demonstrate with TSRS that for the D102N mutant in NaCl (with Na(+) unbound, Na(+) transport) and for WT KR2 in CsCl (with Na(+) unbound, H(+) transport), the extracellular Na(+) binding significantly influences the intermediate K/L/M state equilibrium on the photocycle, while the identity of the transported ion, Na(+) or H(+), does not affect the photocycle. Our findings will contribute to further elucidation of the molecular mechanisms of KR2.
authors.affiliation: Department of Physics and Astronomy, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands. j.t.m.kennis@vu.nl.
authors.forename: Yusaku
authors.initials: Y
authors.lastname: Hontani
authors.forename: Keiichi
authors.initials: K
authors.lastname: Inoue
authors.forename: Miroslav
authors.initials: M
authors.lastname: Kloz
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Kato
authors.forename: Hideki
authors.initials: H
authors.lastname: Kandori
authors.forename: John T M
authors.initials: JT
authors.lastname: Kennis