Rei Abe-Yoshizumi

A light-driven sodium ion pump in marine bacteria

Light-driven proton-pumping rhodopsins are widely distributed in many microorganisms. They convert sunlight energy into proton gradients that serve as energy source of the cell. Here we report a new functional class of a microbial rhodopsin, a light-driven sodium ion pump. We discover that the marine flavobacterium Krokinobacter eikastus possesses two rhodopsins, the first, KR1, being a prototypical proton pump, while the second, KR2, pumps sodium ions outward. Rhodopsin KR2 can also pump lithium ions, but converts to a proton pump when presented with potassium chloride or salts of larger cations. These data indicate that KR2 is a compatible sodium ion-proton pump, and spectroscopic analysis showed it binds sodium ions in its extracellular domain. These findings suggest that light-driven sodium pumps may be as important in situ as their proton-pumping counterparts.

Structural basis for Na + transport mechanism by a light-driven Na + pump

Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na+ pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na+ transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na+ transport. Together with the structure-based engineering of the first light-driven K+ pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

Spectroscopic Study of a Light-Driven Chloride Ion Pump from Marine Bacteria

Thousands of light-driven proton-pumping rhodopsins have been found in marine microbes, and a light-driven sodium-ion pumping rhodopsin was recently discovered, which utilizes sunlight for the energy source of the cell. Similarly, a light-driven chloride-ion pump has been found from marine bacteria, and three eubacterial light-driven pumps possess the DTE (proton pump), NDQ (sodium-ion pump), and NTQ (chloride-ion pump) motifs corresponding to the D85, T89, and D96 positions in bacteriorhodopsin (BR). The corresponding motif of the known haloarchaeal chloride-ion pump, halorhodopsin (HR), is TSA, which is entirely different from the NTQ motif of a eubacterial chloride-ion pump. It is thus intriguing to compare the molecular mechanism of these two chloride-ion pumps. Here we report the spectroscopic study of Fulvimarina rhodopsin (FR), a eubacterial light-driven chloride-ion pump from marine bacterium. FR binds a chloride-ion near the retinal chromophore and chloride-ion binding causes a spectral blue-shift. FR predominantly possesses an all-trans retinal, which is responsible for the light-driven chloride-ion pump. Upon light absorption, the red-shifted K intermediate is formed, followed by the appearance of the L and O intermediates. When the M intermediate does not form, this indicates that the Schiff base remains in the protonated state during the photocycle. These molecular mechanisms are common in HR, and a common mechanism for chloride-ion pumping by evolutionarily distant proteins suggests the importance of the electric quadrupole in the Schiff base region and their changes through hydrogen-bonding alterations. One noticeable difference between FR and HR is the uptake of chloride-ion from the extracellular surface. While the uptake occurs upon decay of the O intermediate in HR, chloride-ion uptake accompanies the rise of the O intermediate in FR. This suggests the presence of a second chloride-ion binding site near the extracellular surface of FR, which is unique to the NTQ rhodopsin.

FTIR Spectroscopy of a Light-Driven Compatible Sodium Ion-Proton Pumping Rhodopsin at 77 K

Krokinobacter eikastus rhodopsin 2 (KR2) is a light-driven sodium ion pump that was discovered in marine bacteria. Although KR2 is able to pump lithium ions similarly, it is converted into a proton pump in potassium chloride or salts of larger cations. In this paper, we applied light-induced difference Fourier-transform infrared (FTIR) spectroscopy to KR2, a compatible sodium ion-proton pump, at 77 K. The first structural study of the functional cycle showed that the structure and structural changes in the primary processes of KR2 are common to all microbial rhodopsins. The red shifted K formation (KR2K) was accompanied by retinal photoisomerization from an all-trans to a 13-cis form, resulting in a distorted retinal chromophore. The observed hydrogen out-of-plane vibrations were H/D exchangeable, indicating that the chromophore distortion by retinal isomerization is located near the Schiff base region in KR2. This tendency was also the case for bacteriorhodopsin and halorhodopsin but not the case for sensory rhodopsin I and II. Therefore, ion pumps such as proton, chloride, and sodium pumps exhibit local structural perturbations of retinal at the Schiff base moiety, while photosensors show more extended structural perturbations of retinal. The retinal Schiff base of KR2 forms a hydrogen bond that is stronger than in BR. KR2 possesses more protein-bound water molecules than other microbial rhodopsins and contains strongly hydrogen-bonded water (O-D stretch at 2333 cm(-1) in D2O). The light-induced difference FTIR spectra at 77 K were identical between the two states functioning as light-driven sodium ion and proton pumps, indicating that the structural changes in the primary processes are identical between different ion pump functions in KR2. In other words, it is unknown which ions are transported by molecules when they absorb photons and photoisomerize. It is likely that the relaxation processes from the K state lead to an alternative function, namely a sodium ion pump or proton pump, depending on the environment.

The Role of the NDQ Motif in Sodium-Pumping Rhodopsins

Sodium-pumping rhodopsins (NaRs) are light-driven outward Na+ pumps. NaRs have a conserved Asn, Asp, and Gln motif (NDQ) in the third transmembrane helix (helix C). The NDQ motif is thus expected to play a crucial role in the operation of the Na+ pump. Herein, we studied the photocycles of the NDQ-motif mutants of Krokinobacter rhodopsin 2 (KR2), the first discovered NaR, by flash photolysis, to obtain insight into the mechanism of Na+ transport. For example, the KR2 N112A mutant did not accumulate the transient red-shifted Na+-bound state, suggesting that Asn112 is vital for the binding of Na+ ions. Additionally, Q123A and Q123V mutants showed significantly slower Na+ uptake and recovery of the initial state. Overall, the Gln123 residue was found to contribute to the optimization of the kinetics of sodium-ion uptake and release. These results demonstrate that the cooperative operation of the three residues of the NDQ motif are important in the operation of the Na+ pump.

Ultrafast Photoreaction Dynamics of a Light-Driven Sodium-Ion-Pumping Retinal Protein from Krokinobacter eikastus Revealed by Femtosecond Time-Resolved Absorption Spectroscopy

We report the first femtosecond time-resolved absorption study on ultrafast photoreaction dynamics of a recently discovered retinal protein, KR2, which functions as a light-driven sodium-ion pump. The obtained data show that the excited-state absorption around 460 nm and the stimulated emission around 720 nm decay concomitantly with a time constant of 180 fs. This demonstrates that the deactivation of the S1 state of KR2, which involves isomerization of the retinal chromophore, takes place three times faster than that of bacteriorhodopsin. In accordance with this rapid electronic relaxation, the photoproduct band assignable to the J intermediate grows up at ∼620 nm, indicating that the J intermediate is directly formed with the S1 → S0 internal conversion. The photoproduct band subsequently exhibits a ∼30 nm blue shift with a 500 fs time constant, corresponding to the conversion to the K intermediate. On the basis of the femtosecond absorption data obtained, we discuss the mechanism for the rapid photoreaction of KR2 and its relevance to the unique function of the sodium-ion pump.

A Color-Determining Amino Acid Residue of Proteorhodopsin

Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria. More than 1000 PRs are classified as blue-absorbing (λmax ∼ 490 nm) and green-absorbing (λmax ∼ 525 nm) PRs. The color determinant is known to be at position 105, where blue-absorbing and green-absorbing PRs possess Gln and Leu, respectively. This suggests hydrophobicity at position 105 plays a key role in color tuning. Here we successfully introduced 19 amino acid residues into position 105 of green-absorbing PR in the membrane environment and investigated the absorption properties. High-performance liquid chromatography analysis shows that the isomeric composition of the all-trans form is >70% for all mutants, indicating little influence of different isomers on color tuning. Absorption spectra of the wild-type and 19 mutant proteins were well-characterized by the pH-dependent equilibria of the protonated and deprotonated counterion (Asp97) of the Schiff base, whereas the λmax values of these two states and the pKa value differed significantly among mutants. Although Gln and Leu are hydrophilic and hydrophobic residues, respectively, the λmax values of the two states and the pKa value did not correlate with the hydropathy index of residues. In contrast, the λmax and pKa were correlated with the volume of residues, though Gln and Leu possess similar volumes. This observation concludes that the λmax and pKa of Asp97 are determined by local and specific interactions in the Schiff base moiety, in which the volume of the residue at position 105 is more influential than its hydrophobicity. We suggest that the hydrogen-bonding network in the Schiff base moiety plays a key role in the λmax and pKa of Asp97, and the hydrogen-bonding network is significantly perturbed by large amino acid residues but may be preserved by additional water molecule(s) for small amino acid residues at position 105.

A Chimera Na+-Pump Rhodopsin as an Effective Optogenetic Silencer

With the progress of optogenetics, the activities of genetically identified neurons can be optically silenced to determine whether the neurons in question are necessary for the network performance of the behavioral expression. This logical induction is expected to be improved by the application of the Na+ pump rhodopsins (NaRs), which hyperpolarize the membrane potential with negligible influence on the ionic/pH balance. Here, we made several chimeric NaRs between two NaRs, KR2 and IaNaR from Krokinobacter eikastus and Indibacter alkaliphilus, respectively. We found that one of these chimeras, named I1K6NaR, exhibited some improvements in the membrane targeting and photocurrent properties over native NaRs. The I1K6NaR-expressing cortical neurons were stably silenced by green light irradiation for a certain long duration. With its rapid kinetics and voltage dependency, the photoactivation of I1K6NaR would specifically counteract the generation of action potentials with less hyperpolarization of the neuronal membrane potential than KR2.

Functional characterization of sodium-pumping rhodopsins with different pumping properties

Sodium pumping rhodopsins (NaRs) are a unique member of the microbial-type I rhodopsin family which actively transport Na+ and H+ depending on ionic condition. In this study, we surveyed 12 different NaRs from various sources of eubacteria for their electrophysiological as well as spectroscopic properties. In mammalian cells several of these NaRs exhibited a Na+ based pump photocurrent and four interesting candidates were chosen for further characterization. Voltage dependent photocurrent amplitudes revealed a membrane potential-sensitive turnover rate, indicating the presence of an electrically-charged intermediate(s) in the photocycle reaction. The NaR from Salinarimonas rosea DSM21201 exhibited a red-shifted absorption spectrum, and slower kinetics compared to the first described sodium pump, KR2. Although the ratio of Na+ to H+ ion transport varied among the NaRs we tested, the NaRs from Flagellimonas sp_DIK and Nonlabens sp_YIK_SED-11 showed significantly higher Na+ selectivity when compared to KR2. All four further investigated NaRs showed a functional expression in dissociated hippocampal neuron culture and hyperpolarizing activity upon light-stimulation. Additionally, all four NaRs allowed optical inhibition of electrically-evoked neuronal spiking. Although efficiency of silencing was 3–5 times lower than silencing with the enhanced version of the proton pump AR3 from Halorubrum sodomense, our data outlines a new approach for hyperpolarization of excitable cells without affecting the intracellular and extracellular proton environment.

Role of Asn112 in a Light-Driven Sodium Ion-Pumping Rhodopsin

Light-driven outward sodium-pumping rhodopsin (NaR) was recently found in marine bacteria. Krokinobacter eikastus rhodopsin 2 (KR2) actively transports sodium and lithium ions in NaCl and LiCl, respectively, while it pumps protons in KCl. NaR has a conserved NDQ (N112, D116, and Q123 in KR2) motif, and previous studies suggested an important role for N112 in the function of KR2. Here we replaced N112 with 19 different amino acids and studied the molecular properties of the mutants. All mutants exhibited absorption bands from a protonated Schiff base in the λmax range from 508 to 531 nm upon heterologous expression in Escherichia coli, whose ion-pumping activity was measured using pH electrodes. The function of these mutants was classified into three phenotypes: wild-type (WT)-like Na+/H+ compatible pump, exclusive H+ pump, and no pump. Among the 19 mutants, only N112D, -G, -S, and -T showed light-driven Na+ pump activity, N112A, -C, -P, -V, -E, -Q, -I, -L, -M, -F, and -W were exclusively H+ pumps, and N112H, -K, -Y, and -R exhibited no pump activity. The mutants of the no pump function lack a blue-shifted M intermediate, indicating that Schiff base deprotonation is a prerequisite for Na+ and H+ pumps. In contrast, the subsequent red-shifted O intermediate was observed for WT and N112V but absent for N112T and N112A, suggesting that observation of this intermediate depends on kinetics. Although N112D, -G, -S, and -T are able to pump Na+, they also pump H+ in NaCl, where Na+ and H+ pumps compete with each other because of the decreased Na+ uptake efficiency. From these facts, an exclusive Na+ pump in NaCl exists only in WT. We conclude that N112 is one of the functional determinants of NaR.