Time-resolved FTIR study of light-driven sodium pump rhodopsins.

Light-driven sodium ion pump rhodopsin (NaR) is a new functional class of microbial rhodopsin. A previous flash photolysis study of Krokinobacter eikastus rhodopsin 2 (KR2) revealed the presence of three kinetically distinct intermediates: K, L/M, and O. Previous low-temperature Fourier-transform infrared (FTIR) spectroscopy of KR2 showed that photoisomerization from the all-trans to the 13-cis form is the primary event of the Na+ pumping photocycle, but structural information on the subsequent intermediates is limited. Here, we applied step-scan time-resolved FTIR spectroscopy to KR2 and Nonlabens dokdonensis rhodopsin 2 (NdR2). Both low-temperature static and time-resolved FTIR spectra resolved a K-like intermediate, and the corresponding spectra showed few differences. Strong hydrogen-out-of-plane (HOOP) vibrations, which appeared in the K intermediate, are common among other rhodopsins. It is, however, unique for NaR that such HOOP bands are persistent in late intermediates, such as L and O intermediates. This observation strongly suggests similar chromophore structures for the K, L, and O intermediates. In fact, an isotope-labeled study that used 12,14-D2 retinal revealed that the chromophore configuration of the O intermediate in NaR is 13-cis. In contrast to the vibrations of the chromophore, those of the protein differ among intermediates, and this is related to the sodium-pumping function. The molecular mechanism of the light-driven sodium pump is discussed on the basis of the present time-resolved FTIR results.

Improved corneal toxicity and permeability of tranilast by the preparation of ophthalmic formulations containing its nanoparticles.

We prepared ophthalmic formulations containing 0.5% tranilast (TL) nanoparticles using 0.005% benzalkonium chloride (BAC), 0.5% D-mannitol, and 2-hydroxypropyl-ß-cyclodextrin (HPßCD), and investigated their usefulness in the ophthalmologic field by evaluating corneal toxicity and permeability. TL nanoparticles were prepared using zirconia beads and Bead Smash 12, which allowed the preparation of high quality dispersions containing 0.5% TL nanoparticles (particle size, 34 ± 20 nm, means ± S.D.). Dispersions containing TL nanoparticles are tolerated better by human corneal epithelium cells than a commercially available 0.5% TL preparation (RIZABEN(®) eye drops). In addition, the addition of TL nanoparticles to the dispersions does not affect the antimicrobial activity of BAC against Escherichia coli (ATCC 8739), and the corneal penetration of TL from dispersions containing TL nanoparticles was significantly higher than in the case of the commercially available 0.5% TL eye drops. It is possible that dispersions containing TL nanoparticles will show increased effectiveness against ocular inflammation, and that ocular drug delivery systems using drug nanoparticles may lead to an expansion of their usefulness for therapy in the ophthalmologic field.

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.