In Vitro Demonstration of Dual Light-Driven Na+/H+ Pumping by a Microbial Rhodopsin

Abstract

A subfamily of rhodopsin pigments was recently discovered in bacteria and proposed to function as dual-function light-driven H(+)/Na(+) pumps, ejecting sodium ions from cells in the presence of sodium and protons in its absence. This proposal was based primarily on light-induced proton flux measurements in suspensions of Escherichia coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the putative Na(+) and H(+) transport definitively. We expressed IAR, an uncharacterized member from Indibacter alkaliphilus in E. coli cell suspensions, and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Na(+) pumping activity in vitro, namely, light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Na(+)-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore. These results meet the criteria for electrogenic Na(+) transport and electrogenic H(+) transport, respectively, in the presence and absence of Na(+). The direction of proton fluxes indicated that IAR was inserted inside-out into our sealed LUV system, which we confirmed by site-directed spin-label electron paramagnetic resonance spectroscopy. We further demonstrate that Na(+) transport by IAR requires Na(+) only on the cytoplasmic side of the protein. The in vitro LUV system proves that the dual light-driven H(+)/Na(+) pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo.

In Vitro Activity of a Purified Natural Anion Channelrhodopsin

“…1). This behavior differs from our previous results on a sodium pump rhodopsin (NaR) in LUVs, which actively transport ions (10). The passive nature of the ion movement in ACR explains the saturation of pH changes after prolonged illumination and the absence of a light-off response.…”

“…Light-induced Channel Activity in LUVs Containing GtACR1-GtACR1-inserted in LUVs were prepared with outwardly directed and inwardly directed chloride gradients. We mon-itored light-induced chloride fluxes by the secondary passive flow of protons (10), in this case caused by the light-induced appearance of chloride conductance. The pH-dependent fluorescent dye pyranine inside the LUV was used to monitor pH changes.…”

Marine Bacterial and Archaeal Ion-Pumping Rhodopsins: Genetic Diversity, Physiology, and Ecology

“…The sodium pumps have been found in Bacteroidetes, as well as in members of the Deinococcus-Thermus phylum isolated from very different environments (40,87,88). Interestingly, sodium pumps have been shown to have dual functionality, transporting protons and halogens depending on the chemical environment (40,50,89). It will be exciting to discern how such variability in function could influence the physiology of bacteria in their natural environment.…”

Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications

“…KR2 contains the “NDQ motif” near the retinylidene Schiff base noted in marine eubacteria (35), so named for their contrast with the DTD and DTE motifs in haloarchaeal proton pumps and proteorhodopsins. More than 10 homologs, termed “NaRs”, have been reported in the literature (11) and functional studies in E. coli cells expressing four different NaRs have been conducted (29, 34, 36–37). KR2 was shown to outwardly pump H + in the absence of Na + in the medium (34) and its pumping of either ion was shown to involve a BR-like outward displacement of helix 6 during the photocycle (38), indicating a close mechanistic relationship to rhodopsin proton pumps.…”

“…Nearly all measurements of Na + and H + transport by NaRs have been conducted by recording passive and active light-driven proton fluxes, respectively, in live cell suspensions of the native organism or heterologously transformed E. coli . One, Ia NaR from Indibacter alkaliphilus (the first two italicized letters indicate the genus and species name of the source organism), has been studied in a purified in vitro unilamellar vesicle system, demonstrating that the dual light-driven H + /Na + pumping functions are intrinsic to the single rhodopsin protein and providing a system in which ion flux measurements are not influenced by bioenergetics processes in living cells (37). …”