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To elucidate the role of extracellular histidines in the modulation of the rat P2X 4 receptor by trace metals, we generated single, double, and triple histidine mutants for residues 140, 241, and 286, replacing them with alanines. cDNAs for the wild-type and receptor mutants were expressed in Xenopus laevis oocytes and in human embryonic kidney 293 cells and examined by the two electrode and patch clamp techniques, respectively. Whereas copper inhibited concentration-dependently the ATP-gated currents in the wild-type and in the single or double H241A and H286A receptor mutants, all receptors containing H140A were insensitive to copper in both cell systems. The characteristic bell-shaped concentration-response curve of zinc observed in the wildtype receptor became sigmoid in both oocytes and human embryonic kidney cells expressing the H140A mutant; in these mutants, the zinc potentiation was 2.5-4-fold larger than in the wild-type. Results with the H140T and H140R mutants further support the importance of a histidine residue at this position. We conclude that His-140 is critical for the action of copper, indicating that this histidine residue, but not His-241 or His-286, forms part of the inhibitory allosteric metal-binding site of the P2X 4 receptor, which is distinct from the putative zinc facilitator binding site.The notion that trace metals such as zinc or copper are atypical brain messengers has attracted much attention in view of their emerging role in the modulation of brain excitability (1). The importance of trace metals in synaptic activity is highlighted by the description that both copper and zinc are stored in synaptic vesicles from where they are released by electrical depolarization, reaching a high micromolar concentration at the synaptic cleft (2-4). Trace metals are known to modulate a wide variety of brain ionotropic receptors such as glycine, N-methyl-D-aspartate, ␥-aminobutyric acid, nicotinic, and the novel nucleotide receptors (P2X) family (5-9). The P2X purinoceptors are homomeric or heteromeric membrane channels gated by extracellular ATP and related synthetic nucleotides; North (10) recently reviewed the principles of the molecular physiology of this family of receptors. Within the P2X receptor family, the P2X 4 is the most widely distributed in the central nervous system, including the cerebellum and the CA1 region of the hippocampus, where it has been proposed to play a role in glutamatergic synapses (11).The P2X 4 receptor is an interesting model of an ionic channel differentially modulated by trace metals. Acuñ a-Castillo et al. (12) and Coddou et al. (13) reported that zinc potentiates the ATP-evoked currents whereas copper has an inhibitory effect on the activity of this receptor. Based on these findings, Acuñ aCastillo et al. (12) proposed that trace metals modulate the activity of the P2X 4 receptor via two separate metal-binding sites. One of these sites has a preferential selectivity for copper and is characterized by a non-competitive inhibition of the ATP-gated channel acti...

Evidence, mostly from experimental models, has accumulated, indicating that modifications of bacterial metabolite concentrations in the large intestine luminal content, notably after changes in the dietary composition, may have important beneficial or deleterious consequences for the colonic epithelial cell metabolism and physiology in terms of mitochondrial energy metabolism, reactive oxygen species production, gene expression, DNA integrity, proliferation, and viability. Recent data suggest that for some bacterial metabolites, like hydrogen sulfide and butyrate, the extent of their oxidation in colonocytes affects their capacity to modulate gene expression in these cells. Modifications of the luminal bacterial metabolite concentrations may, in addition, affect the colonic pH and osmolarity, which are known to affect colonocyte biology per se. Although the colonic epithelium appears able to face, up to some extent, changes in its luminal environment, notably by developing a metabolic adaptive response, some of these modifications may likely affect the homeostatic process of colonic epithelium renewal and the epithelial barrier function. The contribution of major changes in the colonocyte luminal environment in pathological processes, like mucosal inflammation, preneoplasia, and neoplasia, although suggested by several studies, remains to be precisely evaluated, particularly in a long-term perspective.

Umami is the typical taste induced by monosodium glutamate (MSG), which is thought to be detected by the heterodimeric G protein-coupled receptor, T1R1 and T1R3. Previously, we showed that MSG detection thresholds differ substantially between individuals and we further showed that nontaster and hypotaster subjects are associated with nonsynonymous single polymorphisms occurring in the T1R1 and T1R3 genes. Here, we show using functional expression that both amino acid substitutions (A110V and R507Q) in the N-terminal ligand-binding domain of T1R1 and the 2 other ones (F749S and R757C), located in the transmembrane domain of T1R3, severely impair in vitro T1R1/T1R3 response to MSG. A molecular model of the ligand-binding region of T1R1/T1R3 provides a mechanistic explanation supporting functional expression data. The data presented here support causal relations between the genotype and previous in vivo psychophysical studies in human evaluating sensitivity to MSG.

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