Michel Vivaudou - Academia.edu (original) (raw)
Papers by Michel Vivaudou
J Biol Chem, 1997
In heart, G-protein-activated channels are complexes of two homologous proteins, GIRK1 and GIRK4.... more In heart, G-protein-activated channels are complexes of two homologous proteins, GIRK1 and GIRK4. Expression of either protein alone results in barely active or non-active channels, making it difficult to assess the individual contribution of each subunit to the channel complex. The residue Phe 137 , located within the H5 region of GIRK1, is critical to the synergy between GIRK1 and GIRK4 (Chan, K. W., Sui, J. L., Vivaudou, M., and Logothetis, D. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14193-14198). By modifying this residue or the matching residue of GIRK4, Ser 143 , we have been able to generate mutant proteins that produced large inwardly rectifying, G-protein-modulated currents when expressed alone in Xenopus oocytes. The enhanced activity of the heterologous expression of each of two active mutants, GIRK1(F137S) and GIRK4(S143T), was not caused by association with an endogenous oocyte channel subunit, and these mutants did not display apparent differences in the ability to localize to the cell surface compared with their wild-type counterparts. When these functional mutant channels were compared individually with wild-type heteromeric channels, they responded with only small differences to a number of maneuvers involving coexpression with muscarinic receptors, G-protein ␥ subunits, wild-type or mutated G-protein ␣ subunits, and active protomers of pertussis toxin. These experiments, which confirmed the crucial, though not exclusive, role of G␥ in regulating channel activity, demonstrated that GIRK1(F137S) and GIRK4(S143T), and by extrapolation their wild-type counterparts, interact in a qualitatively similar way with G-protein subunits. These findings suggest that functionally important sites of interaction with G-proteins are likely to be located within the homologous regions of GIRK1 and GIRK4 rather than within the divergent terminal regions. They also raise the question of the functional advantage of a heteromeric over homomeric design for G-protein-gated channels.
Biochimica Et Biophysica Acta Biomembranes, Oct 31, 2007
The sulfonylurea glibenclamide is widely used as an open-channel blocker of the CFTR chloride cha... more The sulfonylurea glibenclamide is widely used as an open-channel blocker of the CFTR chloride channel. Here, we used site-directed mutagenesis to identify glibenclamide site of interaction: a positively charged residue K978, located in the cytoplasmic loop 3. Chargeneutralizing mutations K978A, K978Q, K978S abolished the inhibition of forskolin-activated CFTR chloride current by glibenclamide but not by CFTR inh -172. The charge-conservative mutation K978R did not alter glibenclamide sensitivity of CFTR current. Mutations of the neighbouring R975 (R975A, R975S, R975Q) did not affect electrophysiological and pharmacological properties of CFTR. No alteration of halide selectivity was observed with any of these CFTR mutant channels. This study identifies a novel potential inhibitor site within the CFTR molecule, and suggests a novel role of cytoplasmic loop three, within the second transmembrane domain of CFTR protein. This work is the first to report on the role of a residue in a cytoplasmic loop in the mechanism of action of the channel blocker glibenclamide.
J Membrane Biol, 1993
The patch-clamp technique was used to examine the action of intracellular magnesium ions and ADP ... more The patch-clamp technique was used to examine the action of intracellular magnesium ions and ADP in the absence of ATP on skeletal muscle ATP-sensitive potassium channels (K-ATP channels). Inside-out patches were excised from the membrane of sarcolemmal hlebs which arise spontaneously without enzymatic treatment after a frog muscle fiber is split in half.
J Biol Chem, 1997
Heterologous coexpression of recombinant, G protein-gated, inwardly rectifying K+ (GIRK) channel ... more Heterologous coexpression of recombinant, G protein-gated, inwardly rectifying K+ (GIRK) channel subunits has yielded large currents, severalfold greater than those obtained from expression of the individual subunits. Such current enhancement has been obtained from coexpression of the inactive GIRK1 subunit with the low activity GIRK2-5 subunits in Xenopus oocytes. Using deletion and chimeric constructs, we now report the identification of a C-terminal region unique to GIRK1 and a larger central region of GIRK4 highly homologous to GIRK1, both of which are critical for production of large currents. Chimeras containing these two regions produced homomeric channels, exhibiting currents severalfold greater than those from either wild-type subunit alone. G protein regulation of such chimeric channel currents resembled that of wild-type currents. Green fluorescent protein-tagged channels showed that the amount of chimeric channel expressed on the oocyte cell surface was similar to its wild-type counterpart, suggesting that the enhanced activity was not due to differences in relative levels of expression but rather to the coexistence of the chimeric regions. Single-channel recordings of the active chimeras exhibited patterns of activities with open-time kinetics and conductance characteristics representative of those of GIRK4, indicating that the presence of the GIRK1 C-terminal region caused an increase in the frequency of channel openings without affecting their duration.
Pflugers Archiv European Journal of Physiology, Apr 1, 1991
Single-channel currents were recorded from two classes of Ca2+ channels in visceral smooth muscle... more Single-channel currents were recorded from two classes of Ca2+ channels in visceral smooth muscle cells isolated from the stomach of the toad, Bufo marinus: a class of small-conductance channels (approximately 11 pS) and a class of large-conductance channels (approximately 26 pS). Small-conductance channels were present in a majority of patches and gave rise to a slowly inactivating current (t1/2 approximately 250 ms at 0 mV). Openings of large-conductance channels could be unequivocally resolved only in the presence of the dihydropyridine Ca2+ agonist Bay K 8644. Two subtypes of the large-conductance channels were found--those with a very slow rate of decay (greater than 500 ms) and those with a faster one (less than 100 ms). Large-conductance channels resemble L-type Ca2+ channels of other preparations. Small-conductance channels do not fit unambiguously into the other existing categories (i.e., N or T). Correspondence between single-channel and macroscopic Ca2+ currents is discussed.
Bioinformatics, Jul 1, 2007
The Faseb Journal, Jun 1, 1988
recordings from freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus... more recordings from freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus revealed two types of Ca24 currents.
The Journal of General Physiology
The molecular mechanisms underlying the actions of K channel openers (KCOs) on KATe channels were... more The molecular mechanisms underlying the actions of K channel openers (KCOs) on KATe channels were studied with the patch clamp technique in excised inside-out patches from frog skeletal muscle fibers. Benzopyran KCOs (levcromakalim and SR 47063) opened channels partially blocked by ATP, ADP, or ATP~s, with and without Mg 2+, but they had no effects in the absence of internal nucleotides, even after channel activity had significantly declined because of rundown. The effects of KCOs could therefore be attributed solely to a competitive interaction between KCOs and nucleotides, as confirmed by observations that ATP decreased the apparent affinity for KCOs and that, conversely, KCOs decreased ATP or ADP sensitivity. Protons antagonized the action of the non-benzopyran KCOs, pinacidil and aprikalim, by enhancing their dissociation rate. This effect resembled the effect of acidification on benzopyran KCOs (Forestier, C., Y. Depresle, and M. Vivaudou. FEBS Lett. 325:276-280, 1993), suggesting that, in spite of their structural diversity, KCOs could act through the same binding sites. Detailed analysis of the inhibitory effects of protons on channel activity induced by levcromakalim or SR 47063 revealed that, in the presence of 100 p~M ATP, this effect developed steeply between pH 7 and 6 and was half maximal at pH 6.6. These results are in quantitative agreement with an allosteric model of the KAxp channel possessing four protonation sites, two nucleotidic sites accessible preferentially to Mg2+-free nucleotides, and one benzopyran KCO site. The structural implications of this model are discussed.
The American journal of physiology
ABSTRACT
Physiological Reports, 2015
ATP-sensitive potassium channels (K-ATP channels) play a key role in adjusting the membrane poten... more ATP-sensitive potassium channels (K-ATP channels) play a key role in adjusting the membrane potential to the metabolic state of cells. They result from the unique combination of two proteins: the sulfonylurea receptor (SUR), an ATP-binding cassette (ABC) protein, and the inward rectifier K(+) channel Kir6.2. Both subunits associate to form a heterooctamer (4 SUR/4 Kir6.2). SUR modulates channel gating in response to the binding of nucleotides or drugs and Kir6.2 conducts potassium ions. The activity of K-ATP channels varies with their localization. In pancreatic β-cells, SUR1/Kir6.2 channels are partly active at rest while in cardiomyocytes SUR2A/Kir6.2 channels are mostly closed. This divergence of function could be related to differences in the interaction of SUR1 and SUR2A with Kir6.2. Three residues (E1305, I1310, L1313) located in the linker region between transmembrane domain 2 and nucleotide-binding domain 2 of SUR2A were previously found to be involved in the activation pathway linking binding of openers onto SUR2A and channel opening. To determine the role of the equivalent residues in the SUR1 isoform, we designed chimeras between SUR1 and the ABC transporter multidrug resistance-associated protein 1 (MRP1), and used patch clamp recordings on Xenopus oocytes to assess the functionality of SUR1/MRP1 chimeric K-ATP channels. Our results reveal that the same residues in SUR1 and SUR2A are involved in the functional association with Kir6.2, but they display unexpected side-chain specificities which could account for the contrasted properties of pancreatic and cardiac K-ATP channels.
Molecular Pharmacology
ATP-sensitive K(+) (K(ATP)) channels are a complex of an ATP-binding cassette transporter, the su... more ATP-sensitive K(+) (K(ATP)) channels are a complex of an ATP-binding cassette transporter, the sulfonylurea receptor (SUR), and an inward rectifier K(+) channel subunit, Kir6.2. The diverse pharmacological responsiveness of K(ATP) channels from various tissues are thought to arise from distinct SUR isoforms. Thus, when assembled with Kir6. 2, the pancreatic beta cell isoform SUR1 is activated by the hyperglycemic drug diazoxide but not by hypotensive drugs like cromakalim, whereas the cardiac muscle isoform SUR2A is activated by cromakalim and not by diazoxide. We exploited these differences between SUR1 and SUR2A to pursue a chimeric approach designed to identify the structural determinants of SUR involved in the pharmacological activation of K(ATP) channels. Wild-type and chimeric SUR were coexpressed with Kir6.2 in Xenopus oocytes, and we studied the resulting channels with the patch-clamp technique in the excised inside-out configuration. The third transmembrane domain of SUR is found to be an important determinant of the response to cromakalim, which possibly harbors at least part of its binding site. Contrary to expectations, diazoxide sensitivity could not be linked specifically to the carboxyl-terminal end (nucleotide-binding domain 2) of SUR but appeared to involve complex allosteric interactions between transmembrane and nucleotide-binding domains. In addition to providing direct evidence for the structure-function relationship governing K(ATP) channel activation by potassium channel-opening drugs, a family of drugs of the highest therapeutic interest, these findings delineate the determinants of ligand specificity within the modular ATP-binding cassette-transporter architecture of SUR.
Potassium Channels in Cardiovascular Biology, 2001
Page 307. Chapter 15 Molecular Pharmacology of ATP-Sensitive K+ Channels: How and Why? Andre Terz... more Page 307. Chapter 15 Molecular Pharmacology of ATP-Sensitive K+ Channels: How and Why? Andre Terzic and Michel Vivaudou 1. KATP Channels: From Discovery to Structure ATP-sensitive K+(KATP) channels are recognized ...
Methods in enzymology, 2015
Ion channel-coupled receptor (ICCR) is a recent technology based on the fusion of G protein-coupl... more Ion channel-coupled receptor (ICCR) is a recent technology based on the fusion of G protein-coupled receptors (GPCRs) to an ion channel. Binding of ligands on the GPCR triggers conformational changes of the receptor that are mechanically transmitted to the ion channel gates, generating an electrical signal easily detectable with conventional electrophysiological techniques. ICCRs are heterologously expressed in Xenopus oocytes and offers several advantages such as: (i) real-time recordings on single cells, (ii) standard laboratory environment and inexpensive media for Xenopus oocytes maintenance, (iii) absence of protein purification steps, (iv) sensitivity to agonists and antagonists in concentration-dependent manner, (v) compatibility with a Gi/o protein activation assay based on Kir3.x channels, and (vi) ability to detect receptor activation independently of intracellular effectors. This last characteristic of ICCRs led to the development of a functional assay for G protein-"...
Molecular therapy : the journal of the American Society of Gene Therapy, 2015
Molecular pharmacology, 2005
K(ATP) channel openers are a diverse group of molecules able to activate ATP-sensitive K(+) chann... more K(ATP) channel openers are a diverse group of molecules able to activate ATP-sensitive K(+) channels in a tissue-dependent manner by binding to the channel regulatory subunit, the sulfonylurea receptor (SUR), an ATP-binding cassette protein. Residues crucial to this action were previously identified in the last transmembrane helix of SUR, transmembrane helix 17. This study examined the residue at the most important position, 1253 in the muscle isoform SUR2A and the matching 1290 in the pancreatic/neuronal isoform SUR1 (rat numbering). At this position in either isoform, a threonine enables action of openers, whereas a methionine prohibits it. Using single-point mutagenesis, we have examined the physicochemical basis of this phenomenon and discovered that it relied uniquely on side chain volume and not on shape, polarity, or hydrogen-bonding capacity of the residue. Moreover, the aromatic nature of neighboring residues conserved in SUR1 and SUR2A was found necessary for SUR2A to sust...
J Biol Chem, 1997
In heart, G-protein-activated channels are complexes of two homologous proteins, GIRK1 and GIRK4.... more In heart, G-protein-activated channels are complexes of two homologous proteins, GIRK1 and GIRK4. Expression of either protein alone results in barely active or non-active channels, making it difficult to assess the individual contribution of each subunit to the channel complex. The residue Phe 137 , located within the H5 region of GIRK1, is critical to the synergy between GIRK1 and GIRK4 (Chan, K. W., Sui, J. L., Vivaudou, M., and Logothetis, D. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14193-14198). By modifying this residue or the matching residue of GIRK4, Ser 143 , we have been able to generate mutant proteins that produced large inwardly rectifying, G-protein-modulated currents when expressed alone in Xenopus oocytes. The enhanced activity of the heterologous expression of each of two active mutants, GIRK1(F137S) and GIRK4(S143T), was not caused by association with an endogenous oocyte channel subunit, and these mutants did not display apparent differences in the ability to localize to the cell surface compared with their wild-type counterparts. When these functional mutant channels were compared individually with wild-type heteromeric channels, they responded with only small differences to a number of maneuvers involving coexpression with muscarinic receptors, G-protein ␥ subunits, wild-type or mutated G-protein ␣ subunits, and active protomers of pertussis toxin. These experiments, which confirmed the crucial, though not exclusive, role of G␥ in regulating channel activity, demonstrated that GIRK1(F137S) and GIRK4(S143T), and by extrapolation their wild-type counterparts, interact in a qualitatively similar way with G-protein subunits. These findings suggest that functionally important sites of interaction with G-proteins are likely to be located within the homologous regions of GIRK1 and GIRK4 rather than within the divergent terminal regions. They also raise the question of the functional advantage of a heteromeric over homomeric design for G-protein-gated channels.
Biochimica Et Biophysica Acta Biomembranes, Oct 31, 2007
The sulfonylurea glibenclamide is widely used as an open-channel blocker of the CFTR chloride cha... more The sulfonylurea glibenclamide is widely used as an open-channel blocker of the CFTR chloride channel. Here, we used site-directed mutagenesis to identify glibenclamide site of interaction: a positively charged residue K978, located in the cytoplasmic loop 3. Chargeneutralizing mutations K978A, K978Q, K978S abolished the inhibition of forskolin-activated CFTR chloride current by glibenclamide but not by CFTR inh -172. The charge-conservative mutation K978R did not alter glibenclamide sensitivity of CFTR current. Mutations of the neighbouring R975 (R975A, R975S, R975Q) did not affect electrophysiological and pharmacological properties of CFTR. No alteration of halide selectivity was observed with any of these CFTR mutant channels. This study identifies a novel potential inhibitor site within the CFTR molecule, and suggests a novel role of cytoplasmic loop three, within the second transmembrane domain of CFTR protein. This work is the first to report on the role of a residue in a cytoplasmic loop in the mechanism of action of the channel blocker glibenclamide.
J Membrane Biol, 1993
The patch-clamp technique was used to examine the action of intracellular magnesium ions and ADP ... more The patch-clamp technique was used to examine the action of intracellular magnesium ions and ADP in the absence of ATP on skeletal muscle ATP-sensitive potassium channels (K-ATP channels). Inside-out patches were excised from the membrane of sarcolemmal hlebs which arise spontaneously without enzymatic treatment after a frog muscle fiber is split in half.
J Biol Chem, 1997
Heterologous coexpression of recombinant, G protein-gated, inwardly rectifying K+ (GIRK) channel ... more Heterologous coexpression of recombinant, G protein-gated, inwardly rectifying K+ (GIRK) channel subunits has yielded large currents, severalfold greater than those obtained from expression of the individual subunits. Such current enhancement has been obtained from coexpression of the inactive GIRK1 subunit with the low activity GIRK2-5 subunits in Xenopus oocytes. Using deletion and chimeric constructs, we now report the identification of a C-terminal region unique to GIRK1 and a larger central region of GIRK4 highly homologous to GIRK1, both of which are critical for production of large currents. Chimeras containing these two regions produced homomeric channels, exhibiting currents severalfold greater than those from either wild-type subunit alone. G protein regulation of such chimeric channel currents resembled that of wild-type currents. Green fluorescent protein-tagged channels showed that the amount of chimeric channel expressed on the oocyte cell surface was similar to its wild-type counterpart, suggesting that the enhanced activity was not due to differences in relative levels of expression but rather to the coexistence of the chimeric regions. Single-channel recordings of the active chimeras exhibited patterns of activities with open-time kinetics and conductance characteristics representative of those of GIRK4, indicating that the presence of the GIRK1 C-terminal region caused an increase in the frequency of channel openings without affecting their duration.
Pflugers Archiv European Journal of Physiology, Apr 1, 1991
Single-channel currents were recorded from two classes of Ca2+ channels in visceral smooth muscle... more Single-channel currents were recorded from two classes of Ca2+ channels in visceral smooth muscle cells isolated from the stomach of the toad, Bufo marinus: a class of small-conductance channels (approximately 11 pS) and a class of large-conductance channels (approximately 26 pS). Small-conductance channels were present in a majority of patches and gave rise to a slowly inactivating current (t1/2 approximately 250 ms at 0 mV). Openings of large-conductance channels could be unequivocally resolved only in the presence of the dihydropyridine Ca2+ agonist Bay K 8644. Two subtypes of the large-conductance channels were found--those with a very slow rate of decay (greater than 500 ms) and those with a faster one (less than 100 ms). Large-conductance channels resemble L-type Ca2+ channels of other preparations. Small-conductance channels do not fit unambiguously into the other existing categories (i.e., N or T). Correspondence between single-channel and macroscopic Ca2+ currents is discussed.
Bioinformatics, Jul 1, 2007
The Faseb Journal, Jun 1, 1988
recordings from freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus... more recordings from freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus revealed two types of Ca24 currents.
The Journal of General Physiology
The molecular mechanisms underlying the actions of K channel openers (KCOs) on KATe channels were... more The molecular mechanisms underlying the actions of K channel openers (KCOs) on KATe channels were studied with the patch clamp technique in excised inside-out patches from frog skeletal muscle fibers. Benzopyran KCOs (levcromakalim and SR 47063) opened channels partially blocked by ATP, ADP, or ATP~s, with and without Mg 2+, but they had no effects in the absence of internal nucleotides, even after channel activity had significantly declined because of rundown. The effects of KCOs could therefore be attributed solely to a competitive interaction between KCOs and nucleotides, as confirmed by observations that ATP decreased the apparent affinity for KCOs and that, conversely, KCOs decreased ATP or ADP sensitivity. Protons antagonized the action of the non-benzopyran KCOs, pinacidil and aprikalim, by enhancing their dissociation rate. This effect resembled the effect of acidification on benzopyran KCOs (Forestier, C., Y. Depresle, and M. Vivaudou. FEBS Lett. 325:276-280, 1993), suggesting that, in spite of their structural diversity, KCOs could act through the same binding sites. Detailed analysis of the inhibitory effects of protons on channel activity induced by levcromakalim or SR 47063 revealed that, in the presence of 100 p~M ATP, this effect developed steeply between pH 7 and 6 and was half maximal at pH 6.6. These results are in quantitative agreement with an allosteric model of the KAxp channel possessing four protonation sites, two nucleotidic sites accessible preferentially to Mg2+-free nucleotides, and one benzopyran KCO site. The structural implications of this model are discussed.
The American journal of physiology
ABSTRACT
Physiological Reports, 2015
ATP-sensitive potassium channels (K-ATP channels) play a key role in adjusting the membrane poten... more ATP-sensitive potassium channels (K-ATP channels) play a key role in adjusting the membrane potential to the metabolic state of cells. They result from the unique combination of two proteins: the sulfonylurea receptor (SUR), an ATP-binding cassette (ABC) protein, and the inward rectifier K(+) channel Kir6.2. Both subunits associate to form a heterooctamer (4 SUR/4 Kir6.2). SUR modulates channel gating in response to the binding of nucleotides or drugs and Kir6.2 conducts potassium ions. The activity of K-ATP channels varies with their localization. In pancreatic β-cells, SUR1/Kir6.2 channels are partly active at rest while in cardiomyocytes SUR2A/Kir6.2 channels are mostly closed. This divergence of function could be related to differences in the interaction of SUR1 and SUR2A with Kir6.2. Three residues (E1305, I1310, L1313) located in the linker region between transmembrane domain 2 and nucleotide-binding domain 2 of SUR2A were previously found to be involved in the activation pathway linking binding of openers onto SUR2A and channel opening. To determine the role of the equivalent residues in the SUR1 isoform, we designed chimeras between SUR1 and the ABC transporter multidrug resistance-associated protein 1 (MRP1), and used patch clamp recordings on Xenopus oocytes to assess the functionality of SUR1/MRP1 chimeric K-ATP channels. Our results reveal that the same residues in SUR1 and SUR2A are involved in the functional association with Kir6.2, but they display unexpected side-chain specificities which could account for the contrasted properties of pancreatic and cardiac K-ATP channels.
Molecular Pharmacology
ATP-sensitive K(+) (K(ATP)) channels are a complex of an ATP-binding cassette transporter, the su... more ATP-sensitive K(+) (K(ATP)) channels are a complex of an ATP-binding cassette transporter, the sulfonylurea receptor (SUR), and an inward rectifier K(+) channel subunit, Kir6.2. The diverse pharmacological responsiveness of K(ATP) channels from various tissues are thought to arise from distinct SUR isoforms. Thus, when assembled with Kir6. 2, the pancreatic beta cell isoform SUR1 is activated by the hyperglycemic drug diazoxide but not by hypotensive drugs like cromakalim, whereas the cardiac muscle isoform SUR2A is activated by cromakalim and not by diazoxide. We exploited these differences between SUR1 and SUR2A to pursue a chimeric approach designed to identify the structural determinants of SUR involved in the pharmacological activation of K(ATP) channels. Wild-type and chimeric SUR were coexpressed with Kir6.2 in Xenopus oocytes, and we studied the resulting channels with the patch-clamp technique in the excised inside-out configuration. The third transmembrane domain of SUR is found to be an important determinant of the response to cromakalim, which possibly harbors at least part of its binding site. Contrary to expectations, diazoxide sensitivity could not be linked specifically to the carboxyl-terminal end (nucleotide-binding domain 2) of SUR but appeared to involve complex allosteric interactions between transmembrane and nucleotide-binding domains. In addition to providing direct evidence for the structure-function relationship governing K(ATP) channel activation by potassium channel-opening drugs, a family of drugs of the highest therapeutic interest, these findings delineate the determinants of ligand specificity within the modular ATP-binding cassette-transporter architecture of SUR.
Potassium Channels in Cardiovascular Biology, 2001
Page 307. Chapter 15 Molecular Pharmacology of ATP-Sensitive K+ Channels: How and Why? Andre Terz... more Page 307. Chapter 15 Molecular Pharmacology of ATP-Sensitive K+ Channels: How and Why? Andre Terzic and Michel Vivaudou 1. KATP Channels: From Discovery to Structure ATP-sensitive K+(KATP) channels are recognized ...
Methods in enzymology, 2015
Ion channel-coupled receptor (ICCR) is a recent technology based on the fusion of G protein-coupl... more Ion channel-coupled receptor (ICCR) is a recent technology based on the fusion of G protein-coupled receptors (GPCRs) to an ion channel. Binding of ligands on the GPCR triggers conformational changes of the receptor that are mechanically transmitted to the ion channel gates, generating an electrical signal easily detectable with conventional electrophysiological techniques. ICCRs are heterologously expressed in Xenopus oocytes and offers several advantages such as: (i) real-time recordings on single cells, (ii) standard laboratory environment and inexpensive media for Xenopus oocytes maintenance, (iii) absence of protein purification steps, (iv) sensitivity to agonists and antagonists in concentration-dependent manner, (v) compatibility with a Gi/o protein activation assay based on Kir3.x channels, and (vi) ability to detect receptor activation independently of intracellular effectors. This last characteristic of ICCRs led to the development of a functional assay for G protein-"...
Molecular therapy : the journal of the American Society of Gene Therapy, 2015
Molecular pharmacology, 2005
K(ATP) channel openers are a diverse group of molecules able to activate ATP-sensitive K(+) chann... more K(ATP) channel openers are a diverse group of molecules able to activate ATP-sensitive K(+) channels in a tissue-dependent manner by binding to the channel regulatory subunit, the sulfonylurea receptor (SUR), an ATP-binding cassette protein. Residues crucial to this action were previously identified in the last transmembrane helix of SUR, transmembrane helix 17. This study examined the residue at the most important position, 1253 in the muscle isoform SUR2A and the matching 1290 in the pancreatic/neuronal isoform SUR1 (rat numbering). At this position in either isoform, a threonine enables action of openers, whereas a methionine prohibits it. Using single-point mutagenesis, we have examined the physicochemical basis of this phenomenon and discovered that it relied uniquely on side chain volume and not on shape, polarity, or hydrogen-bonding capacity of the residue. Moreover, the aromatic nature of neighboring residues conserved in SUR1 and SUR2A was found necessary for SUR2A to sust...