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.
We have isolated a novel liver-specific organic anion transporter, LST-1, that is expressed exclusively in the human, rat, and mouse liver. LST-1 is a new gene family located between the organic anion transporter family and prostaglandin transporter. LST-1 transports taurocholate (K m ؍ 13.6 M) in a sodium-independent manner. LST-1 also shows broad substrate specificity. It transports conjugated steroids (dehydroepiandrosterone sulfate, estradiol-17-glucuronide, and estrone-3-sulfate), eicosanoids (prostaglandin E 2 , thromboxane B 2 , leukotriene C 4 , leukotriene E 4 ), and thyroid hormones (thyroxine, K m ؍ 3.0 M and triiodothyronine, K m ؍ 2.7 M), reflecting hepatic multispecificity.LST-1 is probably the most important transporter in human liver for clearance of bile acids and organic anions because hepatic levels of another organic anion transporter, OATP, is very low. This is also the first report of the human molecule that transports thyroid hormones.One of the major function of the liver is the removal of various endogenous and exogenous compounds from the circulation (1, 2). This clearance process involves basolateral membrane transport systems that mediate the hepatocellular uptake of bile acids, organic anions, and organic cations (3, 4). One well studied class of substrates are the bile acids. The uptake of taurocholate is mainly mediated by the Na ϩ /taurocholate cotransporting polypeptide (ntcp) in a Na ϩ -dependent manner (5). The uptake of other bile acids (e.g. cholate) occurs predominantly via a Na ϩ -independent mechanism (2, 4). Some amount of taurocholate is also transported by the Na ϩ -independent mechanism. This Na ϩ -independent carrier system further shows a broad substrate specificity transporting conjugated steroids, cardiac glycosides, and other xenobiotics (4).Initially, the organic anion transporter (oatp) 1 family (oatp1, oatp2, oatp3) was considered to represent the Na ϩ -independent transporting mechanisms in the liver (6 -8). Subsequently, a human cDNA, termed OATP, was isolated (9). However, significant differences were found between human OATP and rat oatp family. First, although the substrate specificities were qualitatively similar, significant differences were found between human OATP-and rat oatp family-mediated initial uptake rates and apparent K m values (10, 11). Second, Northern blot analysis of the human OATP showed considerably high expression in the brain, a pattern that is different from any of the oatp family members. These findings strongly suggest the existence of a different group of organic anion transporters in human liver.Here we report the isolation of a novel human organic anion transporter, termed LST-1, which is expressed exclusively in the liver. When expressed in Xenopus oocytes, many of the functional characteristics of LST-1 were identical to the multispecific transporting mechanisms of human liver. These results suggest that LST-1 is the predominant clearance mechanism of several endogenous and exogenous substrates in human liver. MATERIALS ...
Auditory prostheses can partially restore speech comprehension when hearing fails. Sound coding with current prostheses is based on electrical stimulation of auditory neurons and has limited frequency resolution due to broad current spread within the cochlea. In contrast, optical stimulation can be spatially confined, which may improve frequency resolution. Here, we used animal models to characterize optogenetic stimulation, which is the optical stimulation of neurons genetically engineered to express the light-gated ion channel channelrhodopsin-2 (ChR2).
Two complementary DNAs for the organic anion transporter subtypes oatp2 and oatp3, which transport thyroid hormones as well as taurocholate, were isolated from a rat retina cDNA library. The sequence of oatp2 is identical to that recently reported (Noé , B., Hagenbuch, B., Stieger, B., and Meier, P. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10346 -10350), whereas the sequence of oatp3 is novel. oatp3 consists of 670 amino acid residues and exhibits a structural architecture common to the organic anion transporter family, possessing the 12 putative membrane-spanning segments. Oocytes injected with oatp2 and oatp3 cRNAs showed taurocholate uptake in a saturable manner. The oatp2 and oatp3 cRNAinjected oocytes also showed significant uptake of both thyroxine and triiodothyronine. Northern blot and in situ analyses showed that the oatp2 mRNA was widely expressed in neuronal cells of the central nervous system, especially in the hippocampus, cerebellum, and choroid plexus as well as in the retina and liver. The oatp3 mRNA was highly expressed in the kidney and moderately abundant in the retina. This suggests that oatp2 and oatp3 are multifunctional transporters involved in the transport of thyroid hormones in the brain, retina, liver, and kidney.A homeostatic system controls the fluid environment in the brain and keeps its chemical composition relatively constant compared with that of plasma. One mechanism is the bloodbrain barrier, which selectively transports chemical substances via capillary endothelial cells (1). A second essential component is the choroid plexus (blood-cerebrospinal fluid barrier), which secretes or takes up specific chemical substances (2). Although the presence of specific transporting mechanisms has long been postulated, little is known about their molecular identity. Recent molecular biological studies revealed the organic anion transporter family: the Na ϩ -independent organic anion-transporting polypeptide oatp1 from rat liver, which transports bile acid, bromosulfophthalein (BSP), 1 and conjugated and unconjugated steroid hormones (3, 4); the kidney-specific transporter OAT-K1, which transports methotrexate in the basolateral membrane of renal tubules (5); and the prostaglandin transporter (6). Moreover, physiological studies have suggested the presence of other members of the organic anion transporter family (7). Noé et al. (8) have recently reported that a new organic anion transporter subtype (oatp2) is present in rat brain and liver and that the oatp2-expressed oocytes transported cardiac glycoside as well as taurocholate. However, the endogenous substrate of oatp2 and the regional distribution in the brain have not been revealed.It has been suggested that thyroid hormones are transported into the brain via the blood-brain barrier (9) or via the choroid plexus (10). To reveal this mechanism, we focused on the retina. In the retina, the retinal pigment epithelium is the unique source of transthyretin synthesis, and it serves to transport thyroxine (T4) across the blood-retina barr...
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