Abstract
KR2 from marine bacteria Krokinobacter eikastus is a light-driven Na+ pumping rhodopsin family (NaRs) member that actively transports Na+ and/or H+ depending on the ionic state. We here report electrophysiological studies on KR2 to address ion-transport properties under various electrochemical potentials of Δ[Na+], ΔpH, membrane voltage and light quality, because the contributions of these on the pumping activity were less understood so far. After transient expression of KR2 in mammalian cultured cells (ND7/23 cells), photocurrents were measured by whole-cell patch clamp under various intracellular Na+ and pH conditions. When KR2 was continuously illuminated with LED light, two distinct time constants were obtained depending on the Na+ concentration. KR2 exhibited slow ion transport (τoff of 28 ms) below 1.1 mM NaCl and rapid transport (τoff of 11 ms) above 11 mM NaCl. This indicates distinct transporting kinetics of H+ and Na+. Photocurrent amplitude (current density) depends on the intracellular Na+ concentration, as is expected for a Na+ pump. The M-intermediate in the photocycle of KR2 could be transferred into the dark state without net ion transport by blue light illumination on top of green light. The M intermediate was stabilized by higher membrane voltage. Furthermore, we assessed the optogenetic silencing effect of rat cortical neurons after expressing KR2. Light power dependency revealed that action potential was profoundly inhibited by 1.5 mW/mm2 green light illumination, confirming the ability to apply KR2 as an optogenetics silencer.
Highlights
Microbial rhodopsins are retinal-binding membrane proteins which function as ion-transporters, photo-sensor and light-regulated enzymes [1]
Attempts were made in this study to gain a more precise understanding of the biophysical properties of sodium-pumping rhodopsin and to assess the applicability of KR2 in optogenetics research
This low number was partially due to the low expression level of Na+ pumping rhodopsin family (NaRs) in cultured mammalian cells, making the detailed study of ion transporting properties difficult
Summary
Microbial rhodopsins are retinal-binding membrane proteins which function as ion-transporters, photo-sensor and light-regulated enzymes [1]. NaR is one of the microbial rhodopsins that actively transports Na+ and H+ depending on ionic conditions [2, 3]. Since the discovery of KR2, an NaR first identified from a marine flavobacterium Krokinobacter eikastus, its photochemical properties have been intensively investigated by spectroscopy. Upon photoisomerization of all-trans retinal, KR2 undergoes a photocycle which involves K, L, M and O intermediates with different absorption maxima and different protein conformation. It has been proposed that Na+ is taken up from the cytoplasmic side after the M intermediate forms and released as the O intermediate to the extracellular side [2, 4].
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