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Mechanisms of modulation by internal protons of cyclic nucleotide-gated channels cloned from sensory receptor cells
No full textWe have examined the modulation by internal protons of cyclic nucleotide-gated (CNG) channels cloned from bovine olfactory receptor cells and retinal rods. CNG channels Mere studied in excised inside-out membrane patches from Xenopus laevis oocytes previously injected with the mRNA encoding for the subunit 1 of olfactory or rod channels. Channels were activated by cGMP or cAMP, and currents as a function of cyclic nucleotide concentrations were measured as pH(i) varied between 7.6 and 5.0. Increasing internal proton concentrations caused a partial blockage of the single-channel current, consistent with protonation of a single acidic site with a pK(i) of 4.5-4.7, both in rod and in olfactory CNG channels. Channel gating properties were also affected by internal protons. The open probability at law cyclic nucleotide concentrations was greatly increased by lowering pH(i), and the increase was larger when channels were activated by cAMP than by cGMP. Therefore, internal protons affected both channel permeation and gating properties, causing a reduction in single-channel current and an increase in open probability. These effects are likely to be caused by different titratable groups on the channel
Co-expression of wild-type and mutant olfactory cyclic nucleotide-gated channels: restoration of the native sensitivity to Ca2+ and Mg2+ blockage
No full textEpigallocatechin-3-gallate mobilizes intracellular Ca2+ in prostate cancer cells through combined Ca2+ entry and Ca2+-induced Ca2+ release
No full textAims: To elucidate the mechanism by which (−)-epigallocatechin-3-gallate (EGCG) mediates intracellular Ca2+ increase in androgen-independent prostate cancer (PCa) cells. Main methods: Following exposure to different doses of EGCG, viability of DU145 and PC3 PCa cells was evaluated by MTT assay and the intracellular Ca2+ dynamics by the fluorescent Ca2+ chelator Fura-2. The expression of different channels was investigated by qPCR analysis and sulfhydryl bonds by Ellman's assay. Key findings: EGCG inhibited DU145 and PC3 proliferation with IC50 = 46 and 56 μM, respectively, and induced dose-dependent peaks of internal Ca2+ that were dependent on extracellular Ca2+. The expression of TRPC4 and TRPC6 channels was revealed by qPCR in PC3 cells, but lack of effect by modulators and blockers ruled out an exclusive role for these, as well as for voltage-dependent T-type Ca2+ channels. Application of dithiothreitol and catalase and sulfhydryl (SH) measurements showed that EGCG-induced Ca2+ rise depends on SH oxidation, while the effect of EGTA, dantrolene, and the PLC inhibitor U73122 suggested that EGCG-induced Ca2+ influx acts as a trigger for Ca2+-induced Ca2+ release, involving both ryanodine and IP3 receptors. Different from EGCG, ATP caused a rapid Ca2+ increase, which was independent of external Ca2+, but sensitive to U73122. Significance: EGCG induces an internal Ca2+ increase in PCa cells by a multi-step mechanism. As dysregulation of cytosolic Ca2+ is directly linked to apoptosis in PCa cells, these data confirm the possibility of using EGCG as a synergistic adjuvant in combined therapies for recalcitrant malignancies like androgen-independent PCa
'In situ hybridization techniques for the characterization of chromatin higher order structure and gene expression in mammalian cells'
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