Glutamate transporter coupling to Na,K-ATPase.

Deactivation of glutamatergic signaling in the brain is mediated by glutamate uptake into glia and neurons by glutamate transporters. Glutamate transporters are sodium-dependent proteins that putatively rely indirectly on Na,K-ATPases to generate ion gradients that drive transmitter uptake. Based on anatomical colocalization, mutual sodium dependency, and the inhibitory effects of the Na,K-ATPase inhibitor ouabain on glutamate transporter activity, we postulated that glutamate transporters are directly coupled to Na,K-ATPase and that Na,K-ATPase is an essential modulator of glutamate uptake. Na,K-ATPase was purified from rat cerebellum by tandem anion exchange and ouabain affinity chromatography, and the cohort of associated proteins was characterized by mass spectrometry. The alpha1-alpha 3 subunits of Na,K-ATPase were detected, as were the glutamate transporters GLAST and GLT-1, demonstrating that glutamate transporters copurify with Na,K-ATPases. The link between glutamate transporters and Na,K-ATPase was further established by coimmunoprecipitation and colocalization. Analysis of the regulation of glutamate transporter and Na,K-ATPase activities was assessed using [(3)H]D-aspartate, [(3)H]L-glutamate, and rubidium-86 uptake into synaptosomes and cultured astrocytes. In synaptosomes, ouabain produced a dose-dependent inhibition of glutamate transporter and Na,K-ATPase activities, whereas in astrocytes, ouabain showed a bimodal effect whereby glutamate transporter activity was stimulated at 1 microm ouabain and inhibited at higher concentrations. The effects of protein kinase inhibitors on [(3)H]D-aspartate uptake indicated the selective involvement of Src kinases, which are probably a component of the Na,K-ATPase/glutamate transporter complex. These findings demonstrate that glutamate transporters and Na,K-ATPases are part of the same macromolecular complexes and operate as a functional unit to regulate glutamatergic neurotransmission.

First principle computational study on the full conformational space of L-proline diamides.

Ab initio molecular orbital computations were carried out at three levels of theory: RHF/3-21G, RHF/6-31G(d), and B3LYP/6-31G(d), on four model systems of the amino acid proline, HCO-Pro-NH2 [I], HCO-Pro-NH-Me [II], MeCO-Pro-NH2 [III], and MeCO-Pro-NH-Me [IV], representing a systematic variation in the protecting N- and C-terminal groups. Three previously located backbone conformations, gammaL, epsilonL, and alphaL, were characterized together with two ring-puckered forms syn (gauche+ = g+) or "DOWN" and anti (gauche- = g-) or "UP", as well as trans-trans, trans-cis, cis-trans, and cis-cis peptide bond isomers. The topologies of the conformational potential energy cross-sections (PECS) of the potential energy hypersurfaces (PEHS) for compounds [I]-[IV] were explored and analyzed in terms of potential energy curves (PEC), and HCO-Pro-NH2 [I] was also analyzed in terms of potential energy surfaces (PESs). Thermodynamic functions were also calculated for HCO-Pro-NH2 [I] at the CBS-4M and G3MP2 levels of theory. The study confirms that the use of the simplest model, compound [I] with P(N) = P(C) = H, along with the RHF/3-21G level of theory, is an acceptable practice for the analysis of peptide models because only minor differences in geometry and stability are observed.

Expression of a truncated secreted form of the mGluR3 subtype of metabotropic glutamate receptor.

In this study, 10 truncated constructs encompassing all or part of the extracellular ligand binding domain of the mGluR3 subtype of metabotropic glutamate receptor were generated, expressed in human embryonic kidney cells, and tested for secretion and binding of the high affinity agonist [(3)H]DCG-IV. The effect of inserting epitope tags into the N or C termini on cell secretion and radioligand binding was also examined. Secretion into the cell culture media was observed for 8 of the 10 truncated receptors and all secreted forms displayed high affinity agonist binding. The highest level of binding was observed in the C-terminal polyhistidine-tagged receptor truncated at serine 507. Reduction and enzymatic deglycosylation of the serine 507 truncated receptor using endoglycosidase H and PNGase F showed that the secreted receptor was a disulfide-linked dimer containing complex oligosaccharides. Pharmacological characterization demonstrated that the truncated receptor showed the same rank order of potency of agonist binding, a relatively small 2-fold decrease in agonist affinity, and a larger 10-fold decrease in affinity for the antagonist LY341495 compared to the full-length membrane-bound receptor. These results define the essential requirements for ligand binding to the extracellular domain of mGluR3 and highlight parameters important for the optimization of receptor expression in mammalian cells.