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Scholze P Nørregaard L Singer EA Freissmuth M Gether U Sitte HH 《The Journal of biological chemistry》2002,277(24):21505-21513
The human dopamine transporter (hDAT) contains an endogenous high affinity Zn2+ binding site with three coordinating residues on its extracellular face (His193, His375, and Glu396). Upon binding to this site, Zn2+ causes inhibition of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) uptake. We investigated the effect of Zn2+ on outward transport by superfusing hDAT-expressing HEK-293 cells preloaded with [3H]MPP+. Although Zn2+ inhibited uptake, Zn2+ facilitated [3H]MPP+ release induced by amphetamine, MPP+, or K+-induced depolarization specifically at hDAT but not at the human serotonin and the norepinephrine transporter (hNET). Mutation of the Zn2+ coordinating residue His(193) to Lys (the corresponding residue in hNET) eliminated the effect of Zn2+ on efflux. Conversely, the reciprocal mutation (K189H) conferred Zn2+ sensitivity to hNET. The intracellular [3H]MPP+ concentration was varied to generate saturation isotherms; these showed that Zn2+ increased V(max) for efflux (rather than K(M-Efflux-intracellular)). Thus, blockage of inward transport by Zn2+ is not due to a simple inhibition of the transporter turnover rate. The observations provide evidence against the model of facilitated exchange-diffusion and support the concept that inward and outward transport represent discrete operational modes of the transporter. In addition, they indicate a physiological role of Zn2+, because Zn2+ also facilitated transport reversal of DAT in rat striatal slices. 相似文献
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The availability of zinc has a regulatory role in the immune system. It can have either pro- or anti-inflammatory effects,
which both seem to be a consequence of a direct interaction of zinc with the cytokine secretion by monocytes. In this review,
the molecular basis for this effect, the interaction of zinc with the signal transduction of monocytes, is discussed. In particular,
zinc seems to activate or inhibit several signaling pathways that interact with the signal transduction of pathogen sensing
receptors, the so-called Toll-like receptors (TLR), which sense pathogen-derived molecular structures and, upon activation,
lead to secretion of pro-inflammatory cytokines. The interaction of zinc with protein tyrosine phosphatases and protein kinase
C, and a direct modulation of lipopolysaccharide binding to its receptor (TLR-4) all result in enhanced cytokine production.
On the other hand, a complex interaction between zinc, NO and cyclic nucleotide signaling, and inhibition of interleukin-1
receptor associated kinase-1, and inhibitor of kappa B kinase all counteract the production of pro-inflammatory cytokines.
A role for the zinc binding protein metallothionein as a regulator for intracellular zinc signaling is discussed. By acting
on all these signaling molecules, the zinc status of monocytes can have a direct effect on inflammation. 相似文献
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Summary The ultrastructural localization of Ca2+, Mg2+-activated ATPase was studied in phytohaemagglutinin activated lymphocytes and in normal unstimulated lymphocytes. Cells, fixed in paraformaldehyde-glutaraldehyde, were incubated in a medium containing 3mm ATP, 5mm CaCl2 and 2.4mm Pb(NO3)2 in 0.1m tris buffer at pH 8.5, the optimum pH for histochemical demonstration of this enzyme. Reaction product was localized i the endoplasmic reticulum, nuclear membrane, Golgi apparatus and mitochondria and on the membrane surrounding large electron-dense bodies. Cytoplasmic vesicles and the plasma membrane were negative. Activity in unstimulated lymphocytes showed a similar localization but the amount of endoplasmic reticulum was much less than in activated lymphocytes.The pH of the medium was critical for the localization of the enzyme. At pH 7.5, the cytoplasmic reaction was almost completely inhibited but a dense precipitate was present on the outer surface of the plasma membrane. The reaction was stimulated by either Ca2+ or Mg2+ and was greatly decreased in the absence of these cations or in the presence ofp-chloromercuribenzoate orN-ethylmaleimide. Oligomycin inhibited selectively the reaction in mitochondria but not the reaction at other sites. While the reaction in mitochondria showed complete substrate specificity, a mild reaction was obtained at the other sites with uridine diphosphate or sodium -glycophosphate as substrate. ATP was, however, the preferential substate. 相似文献
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Postsynaptic densities (PSDs) isolated from porcine cerebral cortices are large aggregates consisting of more than 30 different proteins. Inductively coupled plasma-mass spectrometric analyses revealed that isolated PSDs contained zinc at a concentration of 4.1 nmol per mg protein. Treatment with 8 m urea lead to dissociation of the PSDs into small components and, concomitantly, depletion of most of their bound zinc. After removal of the urea by dialysis, urea-dissociated PSD proteins did not reassemble into aggregates by themselves. Adding ZnCl2 to urea-treated PSD samples resulted in the assembly of urea-dissociated proteins into large aggregates with morphology and protein composition closely resembling those of the original PSDs. Mg2+, Ca2+, Co2+, Cd2+, Cu2+, Mn2+, Fe3+, K+ and Na+ ions at higher concentrations also induced the aggregation of urea-dissociated PSD protein. The structures of the K+-, Na+-, Mg2+- and Ca2+-induced aggregates were distinct from that of the original PSDs. Our results indicate that the structure of the PSD could be disassembled and reassembled under in vitro conditions. They further suggest that Zn2+ ions, by binding to certain zinc-binding proteins, play an important role in the formation and maintenance of the structure of the PSD. 相似文献
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