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Carston R Wagner - One of the best experts on this subject based on the ideXlab platform.
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structure and functional characterization of human histidine triad Nucleotide Binding Protein 1 mutations associated with inherited axonal neuropathy with neuromyotonia
Journal of Molecular Biology, 2018Co-Authors: Rachit Shah, Kimberly M Maize, Barry C Finzel, Harrison T West, Alexander M Strom, Carston R WagnerAbstract:Abstract Inherited peripheral neuropathies are a group of neurodegenerative disorders that clinically affect 1 in 2500 individuals. Recently, genetic mutations in human histidine Nucleotide-Binding Protein 1 (hHint1) have been strongly and most frequently associated with patients suffering from axonal neuropathy with neuromyotonia. However, the correlation between the impact of these mutations on the hHint1 structure, enzymatic activity and in vivo function has remained ambiguous. Here, we provide detailed biochemical characterization of a set of these hHint1 mutations. Our findings indicate that half of the mutations (R37P, G93D and W123*) resulted in a destabilization of the dimeric state and a significant decrease in catalytic activity and HINT1 inhibitor Binding affinity. The H112N mutant was found to be dimeric, but devoid of catalytic activity, due to the loss of the catalytically essential histidine; nevertheless, it exhibited high affinity to AMP and a HINT1 inhibitor. In contrast to the active-site mutants, the catalytic activity and dimeric structure of the surface mutants, C84R and G89V, were found to be similar to the wild-type enzyme. Taken together, our results suggest that the pathophysiology of inherited axonal neuropathy with neuromyotonia can be induced by conversion of HINT1 from a homodimer to monomer, by modification of select surface residues or by a significant reduction of the enzyme's catalytic efficiency.
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a crystal structure based guide to the design of human histidine triad Nucleotide Binding Protein 1 hhint1 activated protides
Molecular Pharmaceutics, 2017Co-Authors: Kimberly M Maize, Rachit Shah, Carston R Wagner, Alexander Strom, Sidath C Kumarapperuma, Andrew Zhou, Barry C FinzelAbstract:Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad Nucleotide Binding Protein 1 (hHint1) to expose the Nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate Binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural principles revealed by ...
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caught before released structural mapping of the reaction trajectory for the sofosbuvir activating enzyme human histidine triad Nucleotide Binding Protein 1 hhint1
Biochemistry, 2017Co-Authors: Rachit Shah, Kimberly M Maize, Xin Zhou, Barry C Finzel, Carston R WagnerAbstract:Human histidine triad Nucleotide Binding Protein 1 (hHint1) is classified as an efficient nucleoside phosphoramidase and acyl-adenosine monophosphate hydrolase. Human Hint1 has been shown to be essential for the metabolic activation of Nucleotide antiviral proNucleotides (i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir. The active site of hHint1 comprises an ensemble of strictly conserved histidines, including nucleophilic His112. To structurally investigate the mechanism of hHint1 catalysis, we have designed and prepared nucleoside thiophosphoramidate substrates that are able to capture the transiently formed nucleotidylated-His112 intermediate (E*) using time-dependent crystallography. Utilizing a catalytically inactive hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme-substrate (ES1) and product (EP2) complexes were also cocrystallized, respectively, thus providing a structural map of the reaction trajectory. On the basis of these observations and the mechanistic necessity of proton transfers, proton inventory studies were carried out. Although we cannot completely exclude the possibility of more than one proton in flight, the results of these studies were consistent with the transfer of a single proton during the formation of the intermediate. Interestingly, structural analysis revealed that the critical proton transfers required for intermediate formation and hydrolysis may be mediated by a conserved active site water channel. Taken together, our results provide mechanistic insights underpinning histidine nucleophilic catalysis in general and hHint1 catalysis, in particular, thus aiding the design of future proTides and the elucidation of the natural function of the Hint family of enzymes.
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kinetic mechanism of human histidine triad Nucleotide Binding Protein 1
Biochemistry, 2013Co-Authors: Xin Zhou, Tsuifen Chou, Brandon E Aubol, Chinju Park, Richard Wolfenden, Joseph A Adams, Carston R WagnerAbstract:Human histidine triad Nucleotide Binding Protein 1 (hHint1) is a member of a ubiquitous and ancient branch of the histidine triad (HIT) Protein superfamily. hHint1 is a homodimeric Protein that catalyzes the hydrolysis of model substrates, phosphoramidate (TpAd) and acyl adenylate (AIPA), with a high efficiency. Recently, catalytically inactive hHint1 has been identified as the cause of inherited peripheral neuropathy (1). We have carried out the first detailed kinetic mechanistic studies of hHint1 and have found that the reaction mechanism is consistent with a double displacement mechanism, in which the active site nucleophile His112 is first adenylylated by the substrate, followed by hydrolysis of the AMP-enzyme intermediate. A transient burst phase followed by a linear phase from the stopped-flow fluorescence assay indicated that enzyme adenylylation was faster than the subsequent intermediate hydrolysis and product release. Solvent viscosity experiments suggested that both chemical transformation and diffusion-sensitive events (product release or Protein conformational change) limit the overall turnover. The catalytic trapping experiments and data simulation indicated that the true koff rate of the final product AMP is unlikely to control the overall kcat. Therefore, a Protein conformational change associated with product release is likely rate limiting. In addition, the rate of Hint1 adenylylation was found to be dependent on two residues with pKas of 6.5 and 8, with the former pKa agreeing well with the NMR titration results for the pKa of the active site nucleophile His112. When compared to the uncatalyzed rates, hHint1 was shown to enhance acyl-AMP and AMP phosphoramidate hydrolysis by 106 to 108- fold. Taken together, our analysis indicates that hHint1 catalyzes the hydrolysis of phosphoramidate and acyl adenylate with high efficiency, through a mechanism that relies on rapid adenylylation of the active residue, His-112, while being partially rate limited by intermediate hydrolysis and product release associated with a conformational change. Given the high sequence homology of Hint Proteins across all kingdoms of life, it is likely that their kinetic and catalytic mechanisms will be similar to those elucidated for hHint1.
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e coli histidine triad Nucleotide Binding Protein 1 echint is a catalytic regulator of d alanine dehydrogenase dada activity in vivo
PLOS ONE, 2011Co-Authors: Sanaa K Bardaweel, Brahma Ghosh, Tsuifen Chou, Michael J Sadowsky, Carston R WagnerAbstract:Histidine triad Nucleotide Binding Proteins (Hints) are highly conserved members of the histidine triad (HIT) Protein superfamily. Hints comprise the most ancient branch of this superfamily and can be found in Archaea, Bacteria, and Eukaryota. Prokaryotic genomes, including a wide diversity of both Gram-negative and Gram-positive bacteria, typically have one Hint gene encoded by hinT (ycfF in E. coli). Despite their ubiquity, the foundational reason for the wide-spread conservation of Hints across all kingdoms of life remains a mystery. In this study, we used a combination of phenotypic screening and complementation analyses with wild-type and hinT knock-out Escherichia coli strains to show that catalytically active ecHinT is required in E. coli for growth on D-alanine as a sole carbon source. We demonstrate that the expression of catalytically active ecHinT is essential for the activity of the enzyme D-alanine dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-alanine catabolic pathway. Site-directed mutagenesis studies revealed that catalytically active C-terminal mutants of ecHinT are unable to activate DadA activity. In addition, we have designed and synthesized the first cell-permeable inhibitor of ecHinT and demonstrated that the wild-type E. coli treated with the inhibitor exhibited the same phenotype observed for the hinT knock-out strain. These results reveal that the catalytic activity and structure of ecHinT is essential for DadA function and therefore alanine metabolism in E. coli. Moreover, they provide the first biochemical evidence linking the catalytic activity of this ubiquitous Protein to the biological function of Hints in Escherichia coli.
Barry C Finzel - One of the best experts on this subject based on the ideXlab platform.
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structure and functional characterization of human histidine triad Nucleotide Binding Protein 1 mutations associated with inherited axonal neuropathy with neuromyotonia
Journal of Molecular Biology, 2018Co-Authors: Rachit Shah, Kimberly M Maize, Barry C Finzel, Harrison T West, Alexander M Strom, Carston R WagnerAbstract:Abstract Inherited peripheral neuropathies are a group of neurodegenerative disorders that clinically affect 1 in 2500 individuals. Recently, genetic mutations in human histidine Nucleotide-Binding Protein 1 (hHint1) have been strongly and most frequently associated with patients suffering from axonal neuropathy with neuromyotonia. However, the correlation between the impact of these mutations on the hHint1 structure, enzymatic activity and in vivo function has remained ambiguous. Here, we provide detailed biochemical characterization of a set of these hHint1 mutations. Our findings indicate that half of the mutations (R37P, G93D and W123*) resulted in a destabilization of the dimeric state and a significant decrease in catalytic activity and HINT1 inhibitor Binding affinity. The H112N mutant was found to be dimeric, but devoid of catalytic activity, due to the loss of the catalytically essential histidine; nevertheless, it exhibited high affinity to AMP and a HINT1 inhibitor. In contrast to the active-site mutants, the catalytic activity and dimeric structure of the surface mutants, C84R and G89V, were found to be similar to the wild-type enzyme. Taken together, our results suggest that the pathophysiology of inherited axonal neuropathy with neuromyotonia can be induced by conversion of HINT1 from a homodimer to monomer, by modification of select surface residues or by a significant reduction of the enzyme's catalytic efficiency.
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a crystal structure based guide to the design of human histidine triad Nucleotide Binding Protein 1 hhint1 activated protides
Molecular Pharmaceutics, 2017Co-Authors: Kimberly M Maize, Rachit Shah, Carston R Wagner, Alexander Strom, Sidath C Kumarapperuma, Andrew Zhou, Barry C FinzelAbstract:Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad Nucleotide Binding Protein 1 (hHint1) to expose the Nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate Binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural principles revealed by ...
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caught before released structural mapping of the reaction trajectory for the sofosbuvir activating enzyme human histidine triad Nucleotide Binding Protein 1 hhint1
Biochemistry, 2017Co-Authors: Rachit Shah, Kimberly M Maize, Xin Zhou, Barry C Finzel, Carston R WagnerAbstract:Human histidine triad Nucleotide Binding Protein 1 (hHint1) is classified as an efficient nucleoside phosphoramidase and acyl-adenosine monophosphate hydrolase. Human Hint1 has been shown to be essential for the metabolic activation of Nucleotide antiviral proNucleotides (i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir. The active site of hHint1 comprises an ensemble of strictly conserved histidines, including nucleophilic His112. To structurally investigate the mechanism of hHint1 catalysis, we have designed and prepared nucleoside thiophosphoramidate substrates that are able to capture the transiently formed nucleotidylated-His112 intermediate (E*) using time-dependent crystallography. Utilizing a catalytically inactive hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme-substrate (ES1) and product (EP2) complexes were also cocrystallized, respectively, thus providing a structural map of the reaction trajectory. On the basis of these observations and the mechanistic necessity of proton transfers, proton inventory studies were carried out. Although we cannot completely exclude the possibility of more than one proton in flight, the results of these studies were consistent with the transfer of a single proton during the formation of the intermediate. Interestingly, structural analysis revealed that the critical proton transfers required for intermediate formation and hydrolysis may be mediated by a conserved active site water channel. Taken together, our results provide mechanistic insights underpinning histidine nucleophilic catalysis in general and hHint1 catalysis, in particular, thus aiding the design of future proTides and the elucidation of the natural function of the Hint family of enzymes.
Rachit Shah - One of the best experts on this subject based on the ideXlab platform.
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structure and functional characterization of human histidine triad Nucleotide Binding Protein 1 mutations associated with inherited axonal neuropathy with neuromyotonia
Journal of Molecular Biology, 2018Co-Authors: Rachit Shah, Kimberly M Maize, Barry C Finzel, Harrison T West, Alexander M Strom, Carston R WagnerAbstract:Abstract Inherited peripheral neuropathies are a group of neurodegenerative disorders that clinically affect 1 in 2500 individuals. Recently, genetic mutations in human histidine Nucleotide-Binding Protein 1 (hHint1) have been strongly and most frequently associated with patients suffering from axonal neuropathy with neuromyotonia. However, the correlation between the impact of these mutations on the hHint1 structure, enzymatic activity and in vivo function has remained ambiguous. Here, we provide detailed biochemical characterization of a set of these hHint1 mutations. Our findings indicate that half of the mutations (R37P, G93D and W123*) resulted in a destabilization of the dimeric state and a significant decrease in catalytic activity and HINT1 inhibitor Binding affinity. The H112N mutant was found to be dimeric, but devoid of catalytic activity, due to the loss of the catalytically essential histidine; nevertheless, it exhibited high affinity to AMP and a HINT1 inhibitor. In contrast to the active-site mutants, the catalytic activity and dimeric structure of the surface mutants, C84R and G89V, were found to be similar to the wild-type enzyme. Taken together, our results suggest that the pathophysiology of inherited axonal neuropathy with neuromyotonia can be induced by conversion of HINT1 from a homodimer to monomer, by modification of select surface residues or by a significant reduction of the enzyme's catalytic efficiency.
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a crystal structure based guide to the design of human histidine triad Nucleotide Binding Protein 1 hhint1 activated protides
Molecular Pharmaceutics, 2017Co-Authors: Kimberly M Maize, Rachit Shah, Carston R Wagner, Alexander Strom, Sidath C Kumarapperuma, Andrew Zhou, Barry C FinzelAbstract:Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad Nucleotide Binding Protein 1 (hHint1) to expose the Nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate Binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural principles revealed by ...
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caught before released structural mapping of the reaction trajectory for the sofosbuvir activating enzyme human histidine triad Nucleotide Binding Protein 1 hhint1
Biochemistry, 2017Co-Authors: Rachit Shah, Kimberly M Maize, Xin Zhou, Barry C Finzel, Carston R WagnerAbstract:Human histidine triad Nucleotide Binding Protein 1 (hHint1) is classified as an efficient nucleoside phosphoramidase and acyl-adenosine monophosphate hydrolase. Human Hint1 has been shown to be essential for the metabolic activation of Nucleotide antiviral proNucleotides (i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir. The active site of hHint1 comprises an ensemble of strictly conserved histidines, including nucleophilic His112. To structurally investigate the mechanism of hHint1 catalysis, we have designed and prepared nucleoside thiophosphoramidate substrates that are able to capture the transiently formed nucleotidylated-His112 intermediate (E*) using time-dependent crystallography. Utilizing a catalytically inactive hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme-substrate (ES1) and product (EP2) complexes were also cocrystallized, respectively, thus providing a structural map of the reaction trajectory. On the basis of these observations and the mechanistic necessity of proton transfers, proton inventory studies were carried out. Although we cannot completely exclude the possibility of more than one proton in flight, the results of these studies were consistent with the transfer of a single proton during the formation of the intermediate. Interestingly, structural analysis revealed that the critical proton transfers required for intermediate formation and hydrolysis may be mediated by a conserved active site water channel. Taken together, our results provide mechanistic insights underpinning histidine nucleophilic catalysis in general and hHint1 catalysis, in particular, thus aiding the design of future proTides and the elucidation of the natural function of the Hint family of enzymes.
Kimberly M Maize - One of the best experts on this subject based on the ideXlab platform.
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structure and functional characterization of human histidine triad Nucleotide Binding Protein 1 mutations associated with inherited axonal neuropathy with neuromyotonia
Journal of Molecular Biology, 2018Co-Authors: Rachit Shah, Kimberly M Maize, Barry C Finzel, Harrison T West, Alexander M Strom, Carston R WagnerAbstract:Abstract Inherited peripheral neuropathies are a group of neurodegenerative disorders that clinically affect 1 in 2500 individuals. Recently, genetic mutations in human histidine Nucleotide-Binding Protein 1 (hHint1) have been strongly and most frequently associated with patients suffering from axonal neuropathy with neuromyotonia. However, the correlation between the impact of these mutations on the hHint1 structure, enzymatic activity and in vivo function has remained ambiguous. Here, we provide detailed biochemical characterization of a set of these hHint1 mutations. Our findings indicate that half of the mutations (R37P, G93D and W123*) resulted in a destabilization of the dimeric state and a significant decrease in catalytic activity and HINT1 inhibitor Binding affinity. The H112N mutant was found to be dimeric, but devoid of catalytic activity, due to the loss of the catalytically essential histidine; nevertheless, it exhibited high affinity to AMP and a HINT1 inhibitor. In contrast to the active-site mutants, the catalytic activity and dimeric structure of the surface mutants, C84R and G89V, were found to be similar to the wild-type enzyme. Taken together, our results suggest that the pathophysiology of inherited axonal neuropathy with neuromyotonia can be induced by conversion of HINT1 from a homodimer to monomer, by modification of select surface residues or by a significant reduction of the enzyme's catalytic efficiency.
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a crystal structure based guide to the design of human histidine triad Nucleotide Binding Protein 1 hhint1 activated protides
Molecular Pharmaceutics, 2017Co-Authors: Kimberly M Maize, Rachit Shah, Carston R Wagner, Alexander Strom, Sidath C Kumarapperuma, Andrew Zhou, Barry C FinzelAbstract:Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad Nucleotide Binding Protein 1 (hHint1) to expose the Nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate Binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural principles revealed by ...
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caught before released structural mapping of the reaction trajectory for the sofosbuvir activating enzyme human histidine triad Nucleotide Binding Protein 1 hhint1
Biochemistry, 2017Co-Authors: Rachit Shah, Kimberly M Maize, Xin Zhou, Barry C Finzel, Carston R WagnerAbstract:Human histidine triad Nucleotide Binding Protein 1 (hHint1) is classified as an efficient nucleoside phosphoramidase and acyl-adenosine monophosphate hydrolase. Human Hint1 has been shown to be essential for the metabolic activation of Nucleotide antiviral proNucleotides (i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir. The active site of hHint1 comprises an ensemble of strictly conserved histidines, including nucleophilic His112. To structurally investigate the mechanism of hHint1 catalysis, we have designed and prepared nucleoside thiophosphoramidate substrates that are able to capture the transiently formed nucleotidylated-His112 intermediate (E*) using time-dependent crystallography. Utilizing a catalytically inactive hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme-substrate (ES1) and product (EP2) complexes were also cocrystallized, respectively, thus providing a structural map of the reaction trajectory. On the basis of these observations and the mechanistic necessity of proton transfers, proton inventory studies were carried out. Although we cannot completely exclude the possibility of more than one proton in flight, the results of these studies were consistent with the transfer of a single proton during the formation of the intermediate. Interestingly, structural analysis revealed that the critical proton transfers required for intermediate formation and hydrolysis may be mediated by a conserved active site water channel. Taken together, our results provide mechanistic insights underpinning histidine nucleophilic catalysis in general and hHint1 catalysis, in particular, thus aiding the design of future proTides and the elucidation of the natural function of the Hint family of enzymes.
Jennifer T Lamberts - One of the best experts on this subject based on the ideXlab platform.
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role of the guanine Nucleotide Binding Protein gαo in the development of morphine tolerance and dependence
Psychopharmacology, 2018Co-Authors: Jennifer T Lamberts, Lisa D Rosenthal, Emily M Jutkiewicz, John R TraynorAbstract:The use of morphine and other opioids for chronic pain is limited by the development of analgesic tolerance and physical dependence. Morphine produces its effects by activating the μ opioid receptor, which couples to Gαi/o-containing heterotrimeric G Proteins. Evidence suggests that the antinociceptive effects of morphine are mediated by Gαo. However, the role of Gαo in the development of morphine tolerance and dependence is unknown. The objective of the study is to evaluate the contribution of Gαo to the development of morphine tolerance and dependence in mice. 129S6 mice lacking one copy of the Gαo gene (Gαo +/−) were administered morphine acutely or chronically. Mice were examined for tolerance to the antinociceptive action of morphine using the 52 °C hot plate as the nociceptive stimulus and for dependence by evaluating the severity of naltrexone-precipitated withdrawal. Wild-type littermates of the Gαo +/− mice were used as controls. Changes in μ receptor number and function were determined in midbrain and hindbrain homogenates using radioligand Binding and μ agonist-stimulated [35S]GTPγS Binding, respectively. Following either acute or chronic morphine treatment, all mice developed antinociceptive tolerance and physical dependence, regardless of genotype. With chronic morphine treatment, Gαo +/− mice developed tolerance faster and displayed more severe naltrexone-precipitated withdrawal in some behaviors than did wild-type littermates. Morphine tolerance was not associated with changes in μ receptor number or function in brain homogenates from either wild-type or Gαo +/− mice. These data suggest that the guanine Nucleotide Binding Protein Gαo offers some protection against the development of morphine tolerance and dependence.
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role of the guanine Nucleotide Binding Protein gαo in the development of morphine tolerance and dependence
Psychopharmacology, 2018Co-Authors: Jennifer T Lamberts, Lisa D Rosenthal, Emily M Jutkiewicz, John R TraynorAbstract:Rationale The use of morphine and other opioids for chronic pain is limited by the development of analgesic tolerance and physical dependence. Morphine produces its effects by activating the μ opioid receptor, which couples to Gαi/o-containing heterotrimeric G Proteins. Evidence suggests that the antinociceptive effects of morphine are mediated by Gαo. However, the role of Gαo in the development of morphine tolerance and dependence is unknown.
