Glutamate transporters are integral membrane proteins that catalyze a thermodynamically uphill uptake of the neurotransmitter glutamate from the synaptic cleft into the cytoplasm of glial and neuronal cells by harnessing the energy of pre-existing electrochemical gradients of ions. The linchpin of the reaction is the conformational transition of the transporters between outward and inward facing states, in which the substrate binding sites are accessible from the extracellular space and the cytoplasm respectively. Here we describe a crystal structure of a double cysteine mutant of a bacterial homologue of glutamate transporters, GltPh, which is trapped in the inward facing state by cysteine cross-linking. Together with the previously determined crystal structure of GltPh in the outward facing state, the structure of the cross-linked mutant allows us to propose a molecular mechanism, by which GltPh and, by analogy, mammalian glutamate transporters, mediate sodium-coupled substrate uptake.

The Na(+)/I(-) symporter (NIS) is an integral plasma membrane glycoprotein that mediates active I(-) transport into the thyroid follicular cells, the first step in thyroid hormone biosynthesis. NIS-mediated thyroidal I(-) transport from the bloodstream to the colloid is a vectorial process made possible by the selective targeting of NIS to the basolateral membrane. NIS also mediates active I(-) transport in other tissues, including salivary glands, gastric mucosa, and lactating mammary gland, in which it translocates I(-) into the milk for thyroid hormone biosynthesis by the nursing newborn. NIS provides the basis for the effective diagnostic and therapeutic management of thyroid cancer and its metastases with radioiodide. NIS research has proceeded at an astounding pace after the 1996 isolation of the rat NIS cDNA, comprising the elucidation of NIS secondary structure and topology, biogenesis and posttranslational modifications, transcriptional and posttranscriptional regulation, electrophysiological analysis, isolation of the human NIS cDNA, and determination of the human NIS genomic organization. Clinically related topics include the analysis of congenital I(-) transport defect-causing NIS mutations and the role of NIS in thyroid cancer. NIS has been transduced into various kinds of cancer cells to render them susceptible to destruction with radioiodide. Most dramatically, the discovery of endogenous NIS expression in more than 80% of human breast cancer samples has raised the possibility that radioiodide may be a valuable novel tool in breast cancer diagnosis and treatment.

The molecular mechanisms underlying the voltage dependence of intercellular channels formed by the family of vertebrate gap junction proteins (connexins) are unknown. All vertebrate gap junctions are sensitive to the voltage difference between the cells, defined as the transjunctional voltage, Vj (refs 1, 2), and most appear to gate by the separate actions of their component hemichannels. The heterotypic Cx32/Cx26 junction displays an unpredicted rectification that was reported to represent a novel Vj dependence created by hemichannel interactions, mediated in part by the first extracellular loop E1 (ref. 9). Here we show that aspects of the rectification of Cx32/Cx26 junctions are explained by opposite gating polarities of the component hemichannels, and that the opposite gating polarity of Cx32 and Cx26 results from a charge difference in a single amino-acid residue located at the second position in the N terminus. We also show that charge substitutions at the border of the first transmembrane (M1) and E1 domains can reverse gating polarity and suppress the effects of a charge substitution at the N terminus. We conclude that the combined actions of residues at the N terminus and M1/E1 border form a charge complex that is probably an integral part of the connexin voltage sensor. A consistent correlation between charge substitution and gating polarity indicates that Cx26 and Cx32 voltage sensors are oppositely charged and that both move towards the cytoplasm upon hemichannel closure.

TSPO translocator proteins bind steroids and porphyrins, and they are implicated in many human diseases, for which they serve as biomarkers and therapeutic targets. TSPOs have tryptophan-rich sequences that are fhighly conserved from bacteria to mammals. We report crystal structures for Bacillus cereus TSPO (BcTSPO) down to 1.7Å resolution, including a complex with the benzodiazepine-like inhibitor PK11195. We also describe BcTSPO-mediated protoporphyrin IX (PpIX) reactions, including catalytic degradation to a previously undescribed heme derivative. We used structure-inspired mutations to investigate reaction mechanisms, and we showed that TSPOs from Xenopus and man have similar PpIX-directed activities. Although TSPOs have been regarded as transporters, the catalytic activity in PpIX degradation suggests physiological importance for TSPOs in protection against oxidative stress.

Sodium and aspartate symporter from Pyrococcus horikoshii, GltPh, is a homologue of the mammalian glutamate transporters, homotrimeric integral membrane proteins controlling the neurotransmitter levels in brain synapses. These transporters function by alternating between outward and inward facing states, in which the substrate binding site is oriented toward the extracellular space and the cytoplasm, respectively. Here we employ double electron-electron resonance (DEER) spectroscopy to probe the structure and the state distribution of the subunits in the trimer within distinct hydrophobic environments of detergent micelles and lipid bilayers. Our experiments reveal a conformational ensemble of protomers sampling the outward and inward facing states with nearly equal probabilities, indicative of comparable energies, and independently of each other. On average, the distributions vary only modestly in detergent and in bilayers, but in several mutants unique conformations are stabilized by the latter.