In Vitro Activity of a Purified Natural Anion Channelrhodopsin.

Natural anion channelrhodopsins (ACRs) recently discovered in cryptophyte algae are the most active rhodopsin channels known. They are of interest both because of their unique natural function of light-gated chloride conductance and because of their unprecedented efficiency of membrane hyperpolarization for optogenetic neuron silencing. Light-induced currents of ACRs have been studied in HEK cells and neurons, but light-gated channel conductance of ACRs in vitro has not been demonstrated. Here we report light-induced chloride channel activity of a purified ACR protein reconstituted in large unilamellar vesicles (LUVs). EPR measurements establish that the channels are inserted uniformly "inside-out" with their cytoplasmic surface facing the medium of the LUV suspension. We show by time-resolved flash spectroscopy that the photochemical reaction cycle of a functional purified ACR from Guillardia theta (GtACR1) in LUVs exhibits similar spectral shifts, indicating similar photocycle intermediates as GtACR1 in detergent micelles. Furthermore, the photocycle rate is dependent on electric potential generated by chloride gradients in the LUVs in the same manner as in voltage-clamped animal cells. We confirm with this system that, in contrast to cation-conducting channelrhodopsins, opening of the channel occurs prior to deprotonation of the Schiff base. However, the photointermediate transitions in the LUVs exhibit faster kinetics. The ACR-incorporated LUVs provide a purified defined system amenable to EPR, optical and vibrational spectroscopy, and fluorescence resonance energy transfer measurements of structural changes of ACRs with the molecules in a demonstrably functional state.

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

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.

Structural changes in the photoactive site of proteorhodopsin during the primary photoreaction.

Proteorhodopsin (PR), found in marine gamma-proteobacteria, is a newly discovered light-driven proton pump similar to bacteriorhodopsin (BR). Because of the widespread distribution of proteobacteria in the worldwide oceanic waters, this pigment may contribute significantly to the global solar energy input in the biosphere. We examined structural changes that occur during the primary photoreaction (PR --> K) of wild-type pigment and two mutants using low-temperature FTIR difference spectroscopy. Several vibrations detected in the 3500-3700 cm(-1) region are assigned on the basis of H(2)O --> H(2)(18)O exchange to the perturbation of one or more internal water molecules. Substitution of the negatively charged Schiff base counterion, Asp97, with the neutral asparagine caused a downshift of the ethylenic (C=C) and Schiff base (C=N) stretching modes, in agreement with the 27 nm red shift of the visible lambda(max). However, this replacement did not alter the normal all-trans to 13-cis isomerization of the chromophore or the environment of the detected water molecule(s). In contrast, substitution of Asn230, which is in a position to interact with the Schiff base, with Ala induces a 5 nm red shift of the visible lambda(max) and alters the PR chromophore structure, its isomerization to K, and the environment of the detected internal water molecules. The combination of FTIR and site-directed mutagenesis establishes that both Asp97 and Asn230 are perturbed during the primary phototransition. The environment of Asn230 is further altered during the thermal decay of K. These results suggest that significant differences exist in the conformational changes which occur in the photoactive sites of proteorhodopsin and bacteriorhodopsin during the primary photoreaction.