ATP-sensitive potassium (KATP) channels are widely expressed and play key roles in many tissues b... more ATP-sensitive potassium (KATP) channels are widely expressed and play key roles in many tissues by coupling metabolic state to membrane excitability. The SUR subunits confer drug and enhanced nucleotide sensitivity to the pore-forming Kir6 subunit, and so information transfer between the subunits must occur. In our previous study, we identified an electrostatic interaction between Kir6 and SUR2 subunits that was key for allosteric information transfer between the regulatory and pore-forming subunit. In this study, we demonstrate a second putative interaction between Kir6.2-D323 and SUR2A-Q1336 using patch clamp electrophysiological recording, where charge swap mutation of the residues on either side of the potential interaction compromise normal channel function. The Kir6.2-D323K mutation gave rise to a constitutively active, glibenclamide and ATP-insensitive KATP complex, further confirming the importance of information transfer between the Kir6 and SUR2 subunits. Sensitivity to mo...
This article may be used for non-commercial purposes in accordance with Wiley Terms and Condition... more This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.
BACKGROUND AND PURPOSE (À)-Englerin A (EA) is a potent cytotoxic agent against renal carcinoma ce... more BACKGROUND AND PURPOSE (À)-Englerin A (EA) is a potent cytotoxic agent against renal carcinoma cells. It achieves its effects by activation of transient receptor potential canonical (TRPC)4/TRPC1 heteromeric channels. It is also an agonist at channels formed by the related protein, TRPC5. Here, we sought an EA analogue, which might enable a better understanding of these effects of EA. EXPERIMENTAL APPROACH An EA analogue, A54, was synthesized by chemical elaboration of EA. The effects of EA and A54 on the activity of human TRPC4 or TRPC5 channels overexpressed on A498 and HEK 293 cells were investigated, firstly, by measuring intracellular Ca 2+ and, secondly, current using whole-cell patch clamp recordings. KEY RESULTS A54 had weak or no agonist activity at endogenous TRPC4/TRPC1 channels in A498 cells or TRPC4 or TRPC5 homomeric channels overexpressed in HEK 293 cells. A54 strongly inhibited EA-mediated activation of TRPC4/TRPC1 or TRPC5 and weakly inhibited activation of TRPC4. Studies of TRPC5 showed that A54 shifted the EA concentration-response curve to the right without changing its slope, consistent with competitive antagonism. In contrast, Gd 3+-activated TRPC5 or sphingosine-1-phosphateactivated TRPC4 channels were not inhibited but potentiated by A54. A54 did not activate TRPC3 channels or affect the activation of these channels by the agonist 1-oleoyl-2-acetyl-sn-glycerol. CONCLUSIONS AND IMPLICATIONS This study has revealed a new tool compound for EA and TRPC1/4/5 channel research, which could be useful for characterizing endogenous TRPC1/4/5 channels and understanding EA-binding sites and their physiological relevance. Abbreviations A498, human renal cell carcinoma cell line 498; A54, analogue 54; EA, (À)-englerin A; Gd 3+ , gadolinium ion; TRPC, transient receptor potential canonical BJP
(-)-Englerin A (EA) is a natural product which has potent cytotoxic effects on renal cell carcino... more (-)-Englerin A (EA) is a natural product which has potent cytotoxic effects on renal cell carcinoma cells and other types of cancer cell but not non-cancer cells. Although selectively cytotoxic to cancer cells, adverse reaction in mice and rats has been suggested. EA is a remarkably potent activator of ion channels formed by Transient Receptor Potential Canonical 4 and 5 proteins (TRPC4 and TRPC5) and TRPC4 is essential for EA-mediated cancer cell cytotoxicity. Here we specifically investigated the relevance of TRPC4 and TRPC5 to the adverse reaction. Injection of EA (2 mg.kg-1 i.p.) adversely affected mice for about 1 hour, manifesting as a marked reduction in locomotor activity, after which they fully recovered. TRPC4 and TRPC5 single knockout mice were partially protected and double knockout mice fully protected. TRPC4/ TRPC5 double knockout mice were also protected against intravenous injection of EA. Importance of TRPC4/TRPC5 channels was further suggested by pre-administration of Compound 31 (Pico145), a potent and selective small-molecule inhibitor of TRPC4/ TRPC5 channels which did not cause adverse reaction itself but prevented adverse reaction to EA. EA was detected in the plasma but not the brain and so peripheral mechanisms were implicated but not identified. The data confirm the existence of adverse reaction to EA in mice and suggest that it depends on a combination of TRPC4 and TRPC5 which therefore overlaps partially with TRPC4-dependent cancer cell cytotoxicity. The underlying nature of the observed adverse reaction to EA, as a consequence of TRPC4/TRPC5 channel activation, remains unclear and warrants further investigation.
Proteins of the TRPC family can form many homo- and heterotetrameric cation channels permeable to... more Proteins of the TRPC family can form many homo- and heterotetrameric cation channels permeable to Na⁺, K⁺ and Ca. In this review, we focus on channels formed by the isoforms TRPC1, TRPC4 and TRPC5. We review evidence for the formation of different TRPC1/4/5 tetramers, give an overview of recently developed small-molecule TRPC1/4/5 activators and inhibitors, highlight examples of biological roles of TRPC1/4/5 channels in different tissues and pathologies, and discuss how high-quality chemical probes of TRPC1/4/5 modulators can be used to understand the involvement of TRPC1/4/5 channels in physiological and pathophysiological processes.
The therapeutic agents that target ATP-sensitive potassium channels ATP-sensitive potassium (K AT... more The therapeutic agents that target ATP-sensitive potassium channels ATP-sensitive potassium (K ATP) channels are a major drug target for the treatment of type-2 diabetes. K ATP channels are ubiquitously expressed and link the metabolic state to electrical excitability. In pancreatic β-cells, K ATP channels are crucial in the regulation of glucose-induced insulin secretion. Also, K ATP channels are involved in the protection against neuronal seizures and ischaemic stress in the heart, brain and in the regulation of vascular smooth muscle tone. Functional K ATP channels are hetero-octamers composed of two subunits, a pore forming Kir6, which is a member of the inwardly rectifying potassium channels family, and a regulatory sulphonylurea receptor (SUR). In response to nucleotides and pharmaceutical agonists and antagonists, SUR allosterically regulates channel gating. The allosteric communication pathways between these two heterologus proteins in K ATP channels are still poorly understood. This review will highlight the therapeutic agents that target K ATP channels and are used to treat diabetes and cardiovascular diseases.
The birth and discovery of electrophysiological science took place in the 18th century laying t... more The birth and discovery of electrophysiological science took place in the 18th century laying the path for our understanding of nerve membrane ionic currents. The pore-forming proteins, ion channels, are involved and play critical roles in very important physiological and pathological processes, such as neuronal signaling and cardiac excitability, therefore, they serve as therapeutic drug targets. The study of physiological, pharmacological and biophysical properties of ion channels can be done by patch clamp, a gold standard and powerful electrophysiological technique. The current review, in addition to highlight and cover the history of electrophysiology, patch clamp (conventional and automated) technique, and different types of ion channels, will also discuss the importance of ion channels in different neurological diseases and disorders. As the field of neuroscience is growing, this manuscript is intended as a guide to help in understanding the importance of ion channels, part...
The concentration of free cytosolic Ca(2+) and the voltage across the plasma membrane are major d... more The concentration of free cytosolic Ca(2+) and the voltage across the plasma membrane are major determinants of cell function. Ca(2+)-permeable non-selective cationic channels are known to regulate these parameters but understanding of these channels remains inadequate. Here we focus on Transient Receptor Potential Canonical 4 and 5 proteins (TRPC4 and TRPC5) which assemble as homomers or heteromerize with TRPC1 to form Ca(2+)-permeable non-selective cationic channels in many mammalian cell types. Multiple roles have been suggested including in epilepsy, innate fear, pain and cardiac remodeling but limitations in tools to probe these channels have restricted progress. A key question is whether we can overcome these limitations and develop tools which are high-quality, reliable, easy to use and readily accessible for all investigators. Here, through chemical synthesis and studies of native and over-expressed channels by Ca(2+) and patch-clamp assays, we describe compound 31 (C31), a ...
Edited by Roger J. Colbran (؊)-Englerin A ((؊)-EA) has a rapid and potent cytotoxic effect on sev... more Edited by Roger J. Colbran (؊)-Englerin A ((؊)-EA) has a rapid and potent cytotoxic effect on several types of cancer cell that is mediated by plasma membrane ion channels containing transient receptor potential canonical 4 (TRPC4) protein. Because these channels are Ca 2؉permeable, it was initially thought that the cytotoxicity arose as a consequence of Ca 2؉ overload. Here we show that this is not the case and that the effect of (؊)-EA is mediated by a heteromer of TRPC4 and TRPC1 proteins. Both TRPC4 and TRPC1 were required for (؊)-EA cytotoxicity; however, although TRPC4 was necessary for the (؊)-EA-evoked Ca 2؉ elevation, TRPC1 was not. TRPC1 either had no role or was a negative regulator of Ca 2؉ entry. By contrast, both TRPC4 and TRPC1 were necessary for monovalent cation entry evoked by (؊)-EA, and (؊)-EA-evoked cell death was dependent upon entry of the monovalent cation Na ؉. We therefore hypothesized that Na ؉ / K ؉-ATPase might act protectively by counteracting the Na ؉ load resulting from sustained Na ؉ entry. Indeed, inhibition of Na ؉ /K ؉-ATPase by ouabain potently and strongly increased (؊)-EA-evoked cytotoxicity. The data suggest that (؊)-EA achieves cancer cell cytotoxicity by inducing sustained Na ؉ entry through heteromeric TRPC1/TRPC4 channels and that the cytotoxic effect of (؊)-EA can be potentiated by Na ؉ /K ؉-ATPase inhibition.
The TRPC5 proteins assemble to create calcium-permeable, non-selective, cationic channels. We sou... more The TRPC5 proteins assemble to create calcium-permeable, non-selective, cationic channels. We sought novel modulators of these channels through studies of natural products. EXPERIMENTAL APPROACH Intracellular calcium measurements and patch clamp recordings were made from cell lines. Compounds were generated by synthetic chemistry. KEY RESULTS Through a screen of natural products used in traditional Chinese medicines, the flavonol galangin was identified as an inhibitor of lanthanide-evoked calcium entry in TRPC5 overexpressing HEK 293 cells (IC 50 0.45 μM). Galangin also inhibited lanthanideevoked TRPC5-mediated current in whole-cell and outside-out patch recordings. In differentiated 3T3-L1 cells, it inhibited constitutive and lanthanide-evoked calcium entry through endogenous TRPC5-containing channels. The related natural flavonols, kaempferol and quercetin were less potent inhibitors of TRPC5. Myricetin and luteolin lacked effect, and apigenin was a stimulator. Based on structure-activity relationship studies with natural and synthetic flavonols, we designed 3,5,7-trihydroxy-2-(2-bromophenyl)-4H-chromen-4-one (AM12), which inhibited lanthanide-evoked TRPC5 activity with an IC 50 of 0.28 μM. AM12 also inhibited TRPC5 activity evoked by the agonist (À)-Englerin A and was effective in excised outside-out membrane patches, suggesting a relatively direct effect. It inhibited TRPC4 channels similarly, but its inhibitory effect on TRPC1-TRPC5 heteromeric channels was weaker. CONCLUSIONS AND IMPLICATIONS The data suggest that galangin (a natural product from the ginger family) is a TRPC5 inhibitor and that other natural and synthetic flavonoids contain antagonist or agonist capabilities at TRPC5 and closely related channels depending on the substitution patterns of both the chromone core and the phenyl ring.
The journal of physiological sciences : JPS, Jan 12, 2015
In the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, lyotropic ani... more In the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, lyotropic anions with high permeability also bind relatively tightly within the pore. However, the location of permeant anion binding sites, as well as their relationship to anion permeability, is not known. We have identified lysine residue K95 as a key determinant of permeant anion binding in the CFTR pore. Lyotropic anion binding affinity is related to the number of positively charged amino acids located in the inner vestibule of the pore. However, mutations that change the number of positive charges in this pore region have minimal effects on anion permeability. In contrast, a mutation at the narrow pore region alters permeability with minimal effects on anion binding. Our results suggest that a localized permeant anion binding site exists in the pore; however, anion binding to this site has little influence over anion permeability. Implications of this work for the mechanisms of anion recognitio...
We have mapped a cytoplasmic interaction between Kir6 and SUR subunits of ATP-sensitive potassium... more We have mapped a cytoplasmic interaction between Kir6 and SUR subunits of ATP-sensitive potassium channels. We identify residues forming a key salt-bridge controlling transfer of allosteric information from agonist/antagonist binding to gating of the channel pore.
Pflügers Archiv - European Journal of Physiology, 2014
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl −... more The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channel comprises 12 transmembrane (TM) α-helices, arranged in 2 symmetrical groups of 6. However, those TMs that line the channel pore are not completely defined. We used patch clamp recording to compare the accessibility of cysteine-reactive reagents to cysteines introduced into different TMs. Several residues in TM11 were accessible to extracellular and/or intracellular cysteine reactive reagents; however, no reactive cysteines were identified in TMs 5 or 11. Two accessible residues in TM11 (T1115C and S1118C) were found to be more readily modified from the extracellular solution in closed channels, but more readily modified from the intracellular solution in open channels, as previously reported for T338C in TM6. However, the effects of mutagenesis at S1118 (TM11) on a range of pore functional properties were relatively minor compared to the large effects of mutagenesis at T338 (TM6). Our results suggest that the CFTR pore is lined by TM11 but not by TM5 or TM7. Comparison with previous works therefore suggests that the pore is lined by TMs 1, 6, 11, and 12, suggesting that the structure of the open channel pore is asymmetric in terms of the contributions of different TMs. Although TMs 6 and 11 appear to undergo similar conformational changes during channel opening and closing, the influence of these two TMs on the functional properties of the narrowest region of the pore is clearly unequal.
K ATP channels are hetero-octamers composed of two subunits; a pore forming Kir6 and a sulphonylu... more K ATP channels are hetero-octamers composed of two subunits; a pore forming Kir6 and a sulphonylurea receptor (SUR). SUR subunits regulate K ATP channel gating allosterically, in response to nucleotides and pharmacological agents. We have previously shown that residue D323 of Kir6.2 is central to interaction with the nucleotide-binding fold 2 of SUR2A (Rubaiy et al, Biophysical J. 2011: 100; P432a). The aim of this study was to assess whether E332, the corresponding residue in Kir6.1, plays an equally important role in Kir6.1/ SUR2A complexes. Wild type Kir6.1/SUR2A channels expressed in HEK293 cells required activation by potassium channel opener pinacidil (EC 50 = 43.90 5 1.28 mM), in the presence of UDP (10 mM) before passing current. Introduction of the single point mutation E332K into full length Kir6.1 caused constitutive opening of Kir6.1/SUR2A channels in the absence of pinacidil. Reinstatement of putative inter-subunit salt bridges by expressing Kir6.1-E332K with charge reversal mutants SUR2A-Q1336E or SUR2A-K1322D failed to restore regulated opening. Channels containing the Kir6.1E332K mutant were also insensitive to block by high concentrations of glibenclamide (100 mM). However, co-expression of Kir6.1-E332K with SUR2A-K1322D restored glibenclamide sensitivity to wild type levels ((IC 50 = 9.1251.12 nM), p < 0.15 versus wild type Kir6.1/SUR2A channel (IC 50 = 6.1451.13 nM). Together, these data suggest a key functional role for inter-subunit salt bridges involving Kir6.1-E332K. Constitutive channel opening on mutation of this residue suggests that Kir6.1E332 and salt bridge(s) formed between it and the SUR subunit are crucial for stabilizing closed states of Kir6.1-containing K ATP channels.
ATP-sensitive potassium (KATP) channels are widely expressed and play key roles in many tissues b... more ATP-sensitive potassium (KATP) channels are widely expressed and play key roles in many tissues by coupling metabolic state to membrane excitability. The SUR subunits confer drug and enhanced nucleotide sensitivity to the pore-forming Kir6 subunit, and so information transfer between the subunits must occur. In our previous study, we identified an electrostatic interaction between Kir6 and SUR2 subunits that was key for allosteric information transfer between the regulatory and pore-forming subunit. In this study, we demonstrate a second putative interaction between Kir6.2-D323 and SUR2A-Q1336 using patch clamp electrophysiological recording, where charge swap mutation of the residues on either side of the potential interaction compromise normal channel function. The Kir6.2-D323K mutation gave rise to a constitutively active, glibenclamide and ATP-insensitive KATP complex, further confirming the importance of information transfer between the Kir6 and SUR2 subunits. Sensitivity to mo...
This article may be used for non-commercial purposes in accordance with Wiley Terms and Condition... more This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.
BACKGROUND AND PURPOSE (À)-Englerin A (EA) is a potent cytotoxic agent against renal carcinoma ce... more BACKGROUND AND PURPOSE (À)-Englerin A (EA) is a potent cytotoxic agent against renal carcinoma cells. It achieves its effects by activation of transient receptor potential canonical (TRPC)4/TRPC1 heteromeric channels. It is also an agonist at channels formed by the related protein, TRPC5. Here, we sought an EA analogue, which might enable a better understanding of these effects of EA. EXPERIMENTAL APPROACH An EA analogue, A54, was synthesized by chemical elaboration of EA. The effects of EA and A54 on the activity of human TRPC4 or TRPC5 channels overexpressed on A498 and HEK 293 cells were investigated, firstly, by measuring intracellular Ca 2+ and, secondly, current using whole-cell patch clamp recordings. KEY RESULTS A54 had weak or no agonist activity at endogenous TRPC4/TRPC1 channels in A498 cells or TRPC4 or TRPC5 homomeric channels overexpressed in HEK 293 cells. A54 strongly inhibited EA-mediated activation of TRPC4/TRPC1 or TRPC5 and weakly inhibited activation of TRPC4. Studies of TRPC5 showed that A54 shifted the EA concentration-response curve to the right without changing its slope, consistent with competitive antagonism. In contrast, Gd 3+-activated TRPC5 or sphingosine-1-phosphateactivated TRPC4 channels were not inhibited but potentiated by A54. A54 did not activate TRPC3 channels or affect the activation of these channels by the agonist 1-oleoyl-2-acetyl-sn-glycerol. CONCLUSIONS AND IMPLICATIONS This study has revealed a new tool compound for EA and TRPC1/4/5 channel research, which could be useful for characterizing endogenous TRPC1/4/5 channels and understanding EA-binding sites and their physiological relevance. Abbreviations A498, human renal cell carcinoma cell line 498; A54, analogue 54; EA, (À)-englerin A; Gd 3+ , gadolinium ion; TRPC, transient receptor potential canonical BJP
(-)-Englerin A (EA) is a natural product which has potent cytotoxic effects on renal cell carcino... more (-)-Englerin A (EA) is a natural product which has potent cytotoxic effects on renal cell carcinoma cells and other types of cancer cell but not non-cancer cells. Although selectively cytotoxic to cancer cells, adverse reaction in mice and rats has been suggested. EA is a remarkably potent activator of ion channels formed by Transient Receptor Potential Canonical 4 and 5 proteins (TRPC4 and TRPC5) and TRPC4 is essential for EA-mediated cancer cell cytotoxicity. Here we specifically investigated the relevance of TRPC4 and TRPC5 to the adverse reaction. Injection of EA (2 mg.kg-1 i.p.) adversely affected mice for about 1 hour, manifesting as a marked reduction in locomotor activity, after which they fully recovered. TRPC4 and TRPC5 single knockout mice were partially protected and double knockout mice fully protected. TRPC4/ TRPC5 double knockout mice were also protected against intravenous injection of EA. Importance of TRPC4/TRPC5 channels was further suggested by pre-administration of Compound 31 (Pico145), a potent and selective small-molecule inhibitor of TRPC4/ TRPC5 channels which did not cause adverse reaction itself but prevented adverse reaction to EA. EA was detected in the plasma but not the brain and so peripheral mechanisms were implicated but not identified. The data confirm the existence of adverse reaction to EA in mice and suggest that it depends on a combination of TRPC4 and TRPC5 which therefore overlaps partially with TRPC4-dependent cancer cell cytotoxicity. The underlying nature of the observed adverse reaction to EA, as a consequence of TRPC4/TRPC5 channel activation, remains unclear and warrants further investigation.
Proteins of the TRPC family can form many homo- and heterotetrameric cation channels permeable to... more Proteins of the TRPC family can form many homo- and heterotetrameric cation channels permeable to Na⁺, K⁺ and Ca. In this review, we focus on channels formed by the isoforms TRPC1, TRPC4 and TRPC5. We review evidence for the formation of different TRPC1/4/5 tetramers, give an overview of recently developed small-molecule TRPC1/4/5 activators and inhibitors, highlight examples of biological roles of TRPC1/4/5 channels in different tissues and pathologies, and discuss how high-quality chemical probes of TRPC1/4/5 modulators can be used to understand the involvement of TRPC1/4/5 channels in physiological and pathophysiological processes.
The therapeutic agents that target ATP-sensitive potassium channels ATP-sensitive potassium (K AT... more The therapeutic agents that target ATP-sensitive potassium channels ATP-sensitive potassium (K ATP) channels are a major drug target for the treatment of type-2 diabetes. K ATP channels are ubiquitously expressed and link the metabolic state to electrical excitability. In pancreatic β-cells, K ATP channels are crucial in the regulation of glucose-induced insulin secretion. Also, K ATP channels are involved in the protection against neuronal seizures and ischaemic stress in the heart, brain and in the regulation of vascular smooth muscle tone. Functional K ATP channels are hetero-octamers composed of two subunits, a pore forming Kir6, which is a member of the inwardly rectifying potassium channels family, and a regulatory sulphonylurea receptor (SUR). In response to nucleotides and pharmaceutical agonists and antagonists, SUR allosterically regulates channel gating. The allosteric communication pathways between these two heterologus proteins in K ATP channels are still poorly understood. This review will highlight the therapeutic agents that target K ATP channels and are used to treat diabetes and cardiovascular diseases.
The birth and discovery of electrophysiological science took place in the 18th century laying t... more The birth and discovery of electrophysiological science took place in the 18th century laying the path for our understanding of nerve membrane ionic currents. The pore-forming proteins, ion channels, are involved and play critical roles in very important physiological and pathological processes, such as neuronal signaling and cardiac excitability, therefore, they serve as therapeutic drug targets. The study of physiological, pharmacological and biophysical properties of ion channels can be done by patch clamp, a gold standard and powerful electrophysiological technique. The current review, in addition to highlight and cover the history of electrophysiology, patch clamp (conventional and automated) technique, and different types of ion channels, will also discuss the importance of ion channels in different neurological diseases and disorders. As the field of neuroscience is growing, this manuscript is intended as a guide to help in understanding the importance of ion channels, part...
The concentration of free cytosolic Ca(2+) and the voltage across the plasma membrane are major d... more The concentration of free cytosolic Ca(2+) and the voltage across the plasma membrane are major determinants of cell function. Ca(2+)-permeable non-selective cationic channels are known to regulate these parameters but understanding of these channels remains inadequate. Here we focus on Transient Receptor Potential Canonical 4 and 5 proteins (TRPC4 and TRPC5) which assemble as homomers or heteromerize with TRPC1 to form Ca(2+)-permeable non-selective cationic channels in many mammalian cell types. Multiple roles have been suggested including in epilepsy, innate fear, pain and cardiac remodeling but limitations in tools to probe these channels have restricted progress. A key question is whether we can overcome these limitations and develop tools which are high-quality, reliable, easy to use and readily accessible for all investigators. Here, through chemical synthesis and studies of native and over-expressed channels by Ca(2+) and patch-clamp assays, we describe compound 31 (C31), a ...
Edited by Roger J. Colbran (؊)-Englerin A ((؊)-EA) has a rapid and potent cytotoxic effect on sev... more Edited by Roger J. Colbran (؊)-Englerin A ((؊)-EA) has a rapid and potent cytotoxic effect on several types of cancer cell that is mediated by plasma membrane ion channels containing transient receptor potential canonical 4 (TRPC4) protein. Because these channels are Ca 2؉permeable, it was initially thought that the cytotoxicity arose as a consequence of Ca 2؉ overload. Here we show that this is not the case and that the effect of (؊)-EA is mediated by a heteromer of TRPC4 and TRPC1 proteins. Both TRPC4 and TRPC1 were required for (؊)-EA cytotoxicity; however, although TRPC4 was necessary for the (؊)-EA-evoked Ca 2؉ elevation, TRPC1 was not. TRPC1 either had no role or was a negative regulator of Ca 2؉ entry. By contrast, both TRPC4 and TRPC1 were necessary for monovalent cation entry evoked by (؊)-EA, and (؊)-EA-evoked cell death was dependent upon entry of the monovalent cation Na ؉. We therefore hypothesized that Na ؉ / K ؉-ATPase might act protectively by counteracting the Na ؉ load resulting from sustained Na ؉ entry. Indeed, inhibition of Na ؉ /K ؉-ATPase by ouabain potently and strongly increased (؊)-EA-evoked cytotoxicity. The data suggest that (؊)-EA achieves cancer cell cytotoxicity by inducing sustained Na ؉ entry through heteromeric TRPC1/TRPC4 channels and that the cytotoxic effect of (؊)-EA can be potentiated by Na ؉ /K ؉-ATPase inhibition.
The TRPC5 proteins assemble to create calcium-permeable, non-selective, cationic channels. We sou... more The TRPC5 proteins assemble to create calcium-permeable, non-selective, cationic channels. We sought novel modulators of these channels through studies of natural products. EXPERIMENTAL APPROACH Intracellular calcium measurements and patch clamp recordings were made from cell lines. Compounds were generated by synthetic chemistry. KEY RESULTS Through a screen of natural products used in traditional Chinese medicines, the flavonol galangin was identified as an inhibitor of lanthanide-evoked calcium entry in TRPC5 overexpressing HEK 293 cells (IC 50 0.45 μM). Galangin also inhibited lanthanideevoked TRPC5-mediated current in whole-cell and outside-out patch recordings. In differentiated 3T3-L1 cells, it inhibited constitutive and lanthanide-evoked calcium entry through endogenous TRPC5-containing channels. The related natural flavonols, kaempferol and quercetin were less potent inhibitors of TRPC5. Myricetin and luteolin lacked effect, and apigenin was a stimulator. Based on structure-activity relationship studies with natural and synthetic flavonols, we designed 3,5,7-trihydroxy-2-(2-bromophenyl)-4H-chromen-4-one (AM12), which inhibited lanthanide-evoked TRPC5 activity with an IC 50 of 0.28 μM. AM12 also inhibited TRPC5 activity evoked by the agonist (À)-Englerin A and was effective in excised outside-out membrane patches, suggesting a relatively direct effect. It inhibited TRPC4 channels similarly, but its inhibitory effect on TRPC1-TRPC5 heteromeric channels was weaker. CONCLUSIONS AND IMPLICATIONS The data suggest that galangin (a natural product from the ginger family) is a TRPC5 inhibitor and that other natural and synthetic flavonoids contain antagonist or agonist capabilities at TRPC5 and closely related channels depending on the substitution patterns of both the chromone core and the phenyl ring.
The journal of physiological sciences : JPS, Jan 12, 2015
In the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, lyotropic ani... more In the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, lyotropic anions with high permeability also bind relatively tightly within the pore. However, the location of permeant anion binding sites, as well as their relationship to anion permeability, is not known. We have identified lysine residue K95 as a key determinant of permeant anion binding in the CFTR pore. Lyotropic anion binding affinity is related to the number of positively charged amino acids located in the inner vestibule of the pore. However, mutations that change the number of positive charges in this pore region have minimal effects on anion permeability. In contrast, a mutation at the narrow pore region alters permeability with minimal effects on anion binding. Our results suggest that a localized permeant anion binding site exists in the pore; however, anion binding to this site has little influence over anion permeability. Implications of this work for the mechanisms of anion recognitio...
We have mapped a cytoplasmic interaction between Kir6 and SUR subunits of ATP-sensitive potassium... more We have mapped a cytoplasmic interaction between Kir6 and SUR subunits of ATP-sensitive potassium channels. We identify residues forming a key salt-bridge controlling transfer of allosteric information from agonist/antagonist binding to gating of the channel pore.
Pflügers Archiv - European Journal of Physiology, 2014
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl −... more The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channel comprises 12 transmembrane (TM) α-helices, arranged in 2 symmetrical groups of 6. However, those TMs that line the channel pore are not completely defined. We used patch clamp recording to compare the accessibility of cysteine-reactive reagents to cysteines introduced into different TMs. Several residues in TM11 were accessible to extracellular and/or intracellular cysteine reactive reagents; however, no reactive cysteines were identified in TMs 5 or 11. Two accessible residues in TM11 (T1115C and S1118C) were found to be more readily modified from the extracellular solution in closed channels, but more readily modified from the intracellular solution in open channels, as previously reported for T338C in TM6. However, the effects of mutagenesis at S1118 (TM11) on a range of pore functional properties were relatively minor compared to the large effects of mutagenesis at T338 (TM6). Our results suggest that the CFTR pore is lined by TM11 but not by TM5 or TM7. Comparison with previous works therefore suggests that the pore is lined by TMs 1, 6, 11, and 12, suggesting that the structure of the open channel pore is asymmetric in terms of the contributions of different TMs. Although TMs 6 and 11 appear to undergo similar conformational changes during channel opening and closing, the influence of these two TMs on the functional properties of the narrowest region of the pore is clearly unequal.
K ATP channels are hetero-octamers composed of two subunits; a pore forming Kir6 and a sulphonylu... more K ATP channels are hetero-octamers composed of two subunits; a pore forming Kir6 and a sulphonylurea receptor (SUR). SUR subunits regulate K ATP channel gating allosterically, in response to nucleotides and pharmacological agents. We have previously shown that residue D323 of Kir6.2 is central to interaction with the nucleotide-binding fold 2 of SUR2A (Rubaiy et al, Biophysical J. 2011: 100; P432a). The aim of this study was to assess whether E332, the corresponding residue in Kir6.1, plays an equally important role in Kir6.1/ SUR2A complexes. Wild type Kir6.1/SUR2A channels expressed in HEK293 cells required activation by potassium channel opener pinacidil (EC 50 = 43.90 5 1.28 mM), in the presence of UDP (10 mM) before passing current. Introduction of the single point mutation E332K into full length Kir6.1 caused constitutive opening of Kir6.1/SUR2A channels in the absence of pinacidil. Reinstatement of putative inter-subunit salt bridges by expressing Kir6.1-E332K with charge reversal mutants SUR2A-Q1336E or SUR2A-K1322D failed to restore regulated opening. Channels containing the Kir6.1E332K mutant were also insensitive to block by high concentrations of glibenclamide (100 mM). However, co-expression of Kir6.1-E332K with SUR2A-K1322D restored glibenclamide sensitivity to wild type levels ((IC 50 = 9.1251.12 nM), p < 0.15 versus wild type Kir6.1/SUR2A channel (IC 50 = 6.1451.13 nM). Together, these data suggest a key functional role for inter-subunit salt bridges involving Kir6.1-E332K. Constitutive channel opening on mutation of this residue suggests that Kir6.1E332 and salt bridge(s) formed between it and the SUR subunit are crucial for stabilizing closed states of Kir6.1-containing K ATP channels.
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