We report on a novel use of electrochemical scanning force microscopy (SFM) for the investigation... more We report on a novel use of electrochemical scanning force microscopy (SFM) for the investigation of morphological modifications occurring in plasma membranes containing voltage-gated ion channels, on membrane potential variation. Membrane patches of Xenopus laevis oocytes microinjected with exogenous KAT1 cRNA, deposited by a stripping method at the surfaceof a derivatized gold film in inside-out configuration, have been imaged by SFM in an electrochemical cell. A potentiostat was used to maintain a desired potential drop across the membrane. Performing imaging at potential values corresponding to open (2120 mV) and closed (120 mV) states for KAT1, morphological differences in localized sample zones were observed. Particularly, crossshaped features involving a significant membrane portion appear around putative channel locations. The reported approach constitutes the first demonstration of an SPM-based experimentaltechnique suitable to investigate the rearrangements occurring to the plasma membrane containing voltage-gated channels on transmembrane potential variation
ABSTRACT There is increasing evidence that toxic metals play a role in diseases of unknown etiolo... more ABSTRACT There is increasing evidence that toxic metals play a role in diseases of unknown etiology including neurodegenerative diseases. Nickel (Ni) is a transition metal that at high doses and in certain forms is toxic to both human and animals. Ni appears to accumulate in several organs, including the brain and, despite difficulties in its determination, concentrations ranging from 0.1 to 100 μM are consistent with those found in serum of acutely and chronically exposed human subjects. Ni neurotoxicology is largely unexplored and toxicological literature is almost restricted to Ni allergenic properties or carcinogenicity of some of its compounds. However, there are evidences that this metal affects different types of neuronal ionic channels with significant specificity. Ni blocks voltage-dependent calcium channels, being mostly effective on Cav3.2 T-type and Cav2.3 R-type, and this effect has been used to discriminate between different calcium channel types. But this metal also modulates the activity of the N-methyl-d-aspartate type of glutamate receptor channel with similar potency (μM range) and in a complex and subunit-dependent manner. In this chapter, we discuss the effect of Ni on these and other ionic channels and its potential involvement in neuronal injury.
AIMS To elucidate the mechanism by which (-)-epigallocatechin-3-gallate (EGCG) mediates intracell... more AIMS To elucidate the mechanism by which (-)-epigallocatechin-3-gallate (EGCG) mediates intracellular Ca 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 PC3 and DU145 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 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.
Cadmium (Cd), a toxic metal that induces apoptosis and necrosis in a variety of cells, accumulate... more Cadmium (Cd), a toxic metal that induces apoptosis and necrosis in a variety of cells, accumulates in pancreas and may be a cause of diabetes in humans. In the insulinoma cells line HIT-T15 (HIT), we measured internal calcium (Ca) and Cd levels by the fluorescent dye Fura-2 and confirm that L-type voltage-dependent calcium channels (VDCC) play a major role in glucose response and represent a pathway of Cd influx in these cells. Therefore we examined the role of VDCC in acute Cd poisoning by comparing its accumulation and cytotoxic effect in HIT cells and in epithelial-like VDCC-free HeLa cells. Cultures were incubated with 10-300 microM Cd for 15 min-6 h. While negligible at the end of the treatment, HIT cell death was evident after 18-24 h, and it was time-, dose- and serum-dependent. Short (< or = 60 min) Cd treatments with lower doses (< or = 100 microM in serum-free medium) induced delayed apoptotic cell death, as demonstrated by DNA fragmentation on agarose gels and segmentation of DAPI-stained nuclei. Longer incubations and/or higher concentrations caused mainly necrosis. The same treatments were largely harmless in HeLa cells, in which neither death nor DNA fragmentation was observed. The Ca antagonist nimodipine was capable to prevent HIT cell death at lower doses of Cd and to restore the apoptotic condition at higher doses, indicating that reduction of Cd flux through VDCC modulates Cd toxicity. These data demonstrate a specific sensitivity to Cd of insulinoma cells that can be significant for pancreatic beta-cell pathology.
Copper (Cu) is an essential metal present in the human brain and released from synaptic vesicles ... more Copper (Cu) is an essential metal present in the human brain and released from synaptic vesicles following neuronal depolarization. Cu is known to reduce the NMDA receptor (NR) current with IC50≈20 µM. We have studied the effect of Cu on the NR current in cultured neonatal rat cerebellum granule cells (CGC) and in transiently transfected HEK293 cells (HEK), expressing either GluN1/GLUN2A or GluN1/GluN2B receptors. In CGCs, Cu causes a potentiation of the NR current at concentrations <30 µM (EC50=4.6 µM) and a block at higher concentrations (IC50=24 µM). In Fura2 loaded CGCs, Cu (≤30 µM) caused an increase of NMDA-driven calcium influx. This facilitating effect was prevented by pre-treatment with the reducing agent DTT. Cu also caused an increase of the NR current in GluN1/GluN2A receptors (EC50=2 µM) and a block at higher concentrations (IC50=26 µM). Both facilitation and inhibition were independent of voltage. The effect of Cu was quantitatively similar in GluN1/GluN2B receptors, which were potentiated by 10 µM and inhibited by 100 µM Cu. Potentiation was absent in mutants deleted of their entire amino terminal domain (ATD) of the protein, suggesting an involvement of this region in the interaction. These results indicate that Cu can facilitate the NR current at lower concentrations than those required for blocking it; this effect can have consequences on the activity of the metal at synaptic and extrasynaptic sites.
Background and purposeThe volume regulated anion channel (VRAC) is known to be involved in differ... more Background and purposeThe volume regulated anion channel (VRAC) is known to be involved in different aspects of cancer cell behaviour and response to therapies. For this reason, we investigated the effect of DCPIB, a presumably specific blocker of VRAC, in two types of cancer: pancreatic duct adenocarcinoma (PDAC) and melanoma.Experimental approachWe used patch‐clamp electrophysiology, supported by Ca2+ imaging, gene expression analysis, docking simulation and mutagenesis. We employed two PDAC lines (Panc‐1 and MiaPaCa‐2), as well as a primary (IGR39) and a metastatic (IGR37) melanoma line.Key resultsDCPIB markedly increased whole‐cell currents in Panc‐1, MiaPaca2 and IGR39, but not in IGR37 cells. The currents were mostly mediated by KCa1.1 channels, commonly known as BK channels. We confirmed DCPIB activation of BK channels also in HEK293 cells transfected with α subunits of this channel. Further experiments showed that in IGR39, and to a smaller degree also in Panc‐1 cells, DCPIB induced a rapid Ca2+ influx. This, in turn, indirectly potentiated BK channels and, in IGR39 cells, additionally activated other Ca2+‐dependent channels. However, Ca2+ influx was not required for activation of BK channels by DCPIB, as such activation involved the extracellular part of the protein and we have identified a residue crucial for binding.Conclusion and implicationsDCPIB directly targeted BK channels and, also, acutely increased intracellular Ca2+. Our findings extend the list of DCPIB effects that should be taken into consideration for future development of DCPIB‐based modulators of ion channels and other membrane proteins.
We report on a novel use of electrochemical scanning force microscopy (SFM) for the investigation... more We report on a novel use of electrochemical scanning force microscopy (SFM) for the investigation of morphological modifications occurring in plasma membranes containing voltage-gated ion channels, on membrane potential variation. Membrane patches of Xenopus laevis oocytes microinjected with exogenous KAT1 cRNA, deposited by a stripping method at the surfaceof a derivatized gold film in inside-out configuration, have been imaged by SFM in an electrochemical cell. A potentiostat was used to maintain a desired potential drop across the membrane. Performing imaging at potential values corresponding to open (2120 mV) and closed (120 mV) states for KAT1, morphological differences in localized sample zones were observed. Particularly, crossshaped features involving a significant membrane portion appear around putative channel locations. The reported approach constitutes the first demonstration of an SPM-based experimentaltechnique suitable to investigate the rearrangements occurring to the plasma membrane containing voltage-gated channels on transmembrane potential variation
ABSTRACT There is increasing evidence that toxic metals play a role in diseases of unknown etiolo... more ABSTRACT There is increasing evidence that toxic metals play a role in diseases of unknown etiology including neurodegenerative diseases. Nickel (Ni) is a transition metal that at high doses and in certain forms is toxic to both human and animals. Ni appears to accumulate in several organs, including the brain and, despite difficulties in its determination, concentrations ranging from 0.1 to 100 μM are consistent with those found in serum of acutely and chronically exposed human subjects. Ni neurotoxicology is largely unexplored and toxicological literature is almost restricted to Ni allergenic properties or carcinogenicity of some of its compounds. However, there are evidences that this metal affects different types of neuronal ionic channels with significant specificity. Ni blocks voltage-dependent calcium channels, being mostly effective on Cav3.2 T-type and Cav2.3 R-type, and this effect has been used to discriminate between different calcium channel types. But this metal also modulates the activity of the N-methyl-d-aspartate type of glutamate receptor channel with similar potency (μM range) and in a complex and subunit-dependent manner. In this chapter, we discuss the effect of Ni on these and other ionic channels and its potential involvement in neuronal injury.
AIMS To elucidate the mechanism by which (-)-epigallocatechin-3-gallate (EGCG) mediates intracell... more AIMS To elucidate the mechanism by which (-)-epigallocatechin-3-gallate (EGCG) mediates intracellular Ca 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 PC3 and DU145 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 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.
Cadmium (Cd), a toxic metal that induces apoptosis and necrosis in a variety of cells, accumulate... more Cadmium (Cd), a toxic metal that induces apoptosis and necrosis in a variety of cells, accumulates in pancreas and may be a cause of diabetes in humans. In the insulinoma cells line HIT-T15 (HIT), we measured internal calcium (Ca) and Cd levels by the fluorescent dye Fura-2 and confirm that L-type voltage-dependent calcium channels (VDCC) play a major role in glucose response and represent a pathway of Cd influx in these cells. Therefore we examined the role of VDCC in acute Cd poisoning by comparing its accumulation and cytotoxic effect in HIT cells and in epithelial-like VDCC-free HeLa cells. Cultures were incubated with 10-300 microM Cd for 15 min-6 h. While negligible at the end of the treatment, HIT cell death was evident after 18-24 h, and it was time-, dose- and serum-dependent. Short (< or = 60 min) Cd treatments with lower doses (< or = 100 microM in serum-free medium) induced delayed apoptotic cell death, as demonstrated by DNA fragmentation on agarose gels and segmentation of DAPI-stained nuclei. Longer incubations and/or higher concentrations caused mainly necrosis. The same treatments were largely harmless in HeLa cells, in which neither death nor DNA fragmentation was observed. The Ca antagonist nimodipine was capable to prevent HIT cell death at lower doses of Cd and to restore the apoptotic condition at higher doses, indicating that reduction of Cd flux through VDCC modulates Cd toxicity. These data demonstrate a specific sensitivity to Cd of insulinoma cells that can be significant for pancreatic beta-cell pathology.
Copper (Cu) is an essential metal present in the human brain and released from synaptic vesicles ... more Copper (Cu) is an essential metal present in the human brain and released from synaptic vesicles following neuronal depolarization. Cu is known to reduce the NMDA receptor (NR) current with IC50≈20 µM. We have studied the effect of Cu on the NR current in cultured neonatal rat cerebellum granule cells (CGC) and in transiently transfected HEK293 cells (HEK), expressing either GluN1/GLUN2A or GluN1/GluN2B receptors. In CGCs, Cu causes a potentiation of the NR current at concentrations <30 µM (EC50=4.6 µM) and a block at higher concentrations (IC50=24 µM). In Fura2 loaded CGCs, Cu (≤30 µM) caused an increase of NMDA-driven calcium influx. This facilitating effect was prevented by pre-treatment with the reducing agent DTT. Cu also caused an increase of the NR current in GluN1/GluN2A receptors (EC50=2 µM) and a block at higher concentrations (IC50=26 µM). Both facilitation and inhibition were independent of voltage. The effect of Cu was quantitatively similar in GluN1/GluN2B receptors, which were potentiated by 10 µM and inhibited by 100 µM Cu. Potentiation was absent in mutants deleted of their entire amino terminal domain (ATD) of the protein, suggesting an involvement of this region in the interaction. These results indicate that Cu can facilitate the NR current at lower concentrations than those required for blocking it; this effect can have consequences on the activity of the metal at synaptic and extrasynaptic sites.
Background and purposeThe volume regulated anion channel (VRAC) is known to be involved in differ... more Background and purposeThe volume regulated anion channel (VRAC) is known to be involved in different aspects of cancer cell behaviour and response to therapies. For this reason, we investigated the effect of DCPIB, a presumably specific blocker of VRAC, in two types of cancer: pancreatic duct adenocarcinoma (PDAC) and melanoma.Experimental approachWe used patch‐clamp electrophysiology, supported by Ca2+ imaging, gene expression analysis, docking simulation and mutagenesis. We employed two PDAC lines (Panc‐1 and MiaPaCa‐2), as well as a primary (IGR39) and a metastatic (IGR37) melanoma line.Key resultsDCPIB markedly increased whole‐cell currents in Panc‐1, MiaPaca2 and IGR39, but not in IGR37 cells. The currents were mostly mediated by KCa1.1 channels, commonly known as BK channels. We confirmed DCPIB activation of BK channels also in HEK293 cells transfected with α subunits of this channel. Further experiments showed that in IGR39, and to a smaller degree also in Panc‐1 cells, DCPIB induced a rapid Ca2+ influx. This, in turn, indirectly potentiated BK channels and, in IGR39 cells, additionally activated other Ca2+‐dependent channels. However, Ca2+ influx was not required for activation of BK channels by DCPIB, as such activation involved the extracellular part of the protein and we have identified a residue crucial for binding.Conclusion and implicationsDCPIB directly targeted BK channels and, also, acutely increased intracellular Ca2+. Our findings extend the list of DCPIB effects that should be taken into consideration for future development of DCPIB‐based modulators of ion channels and other membrane proteins.
Uploads