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Paul Whiteaker - One of the best experts on this subject based on the ideXlab platform.
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differential α4 β2 agonist binding site contributions to α4β2 nicotinic acetylcholine receptor Function within and between isoforms
Journal of Biological Chemistry, 2016Co-Authors: Linda Lucero, Maegan M Weltzin, Brek J Eaton, John F Cooper, Jon Lindstrom, Ronald J Lukas, Paul WhiteakerAbstract:Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and Maximum Function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with (125)I-mAb 295 binding and was used to define Function/nAChR. If the α4(+)/(-)β2 sites contribute equally to Function, making identical β2HQT substitutions at either site should produce similar Functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.
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differential α4 β2 agonist binding site contributions to α4β2 nicotinic acetylcholine receptor Function within and between isoforms
Journal of Biological Chemistry, 2016Co-Authors: Linda Lucero, Maegan M Weltzin, Brek J Eaton, John F Cooper, Jon Lindstrom, Ronald J Lukas, Paul WhiteakerAbstract:Abstract Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries, and high vs. low agonist sensitivities, (HS & LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist binding sites in, and between, the isoforms have not been studied. We used a fully-linked subunit concatemeric nAChR approach to express uniform populations of HS- or LS-isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)face E-loop residues to their non-conserved α4 subunit counterparts, or vice-versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. ACh concentration-response curves and Maximum Function were measured using two-electrode voltage-clamp electrophysiology. Surface expression was measured with [125I]mAb 295 binding, and was used to define Function/nAChR. If the α4(+)/(-)β2 sites contribute equally to Function, making identical β2HQT substitutions at either site should produce similar Functional outcomes. Instead, highly-differential outcomes within the HS-isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise-equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation, and that E-loop residues are an important contributor to this neighbor effect.
Linda Lucero - One of the best experts on this subject based on the ideXlab platform.
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differential α4 β2 agonist binding site contributions to α4β2 nicotinic acetylcholine receptor Function within and between isoforms
Journal of Biological Chemistry, 2016Co-Authors: Linda Lucero, Maegan M Weltzin, Brek J Eaton, John F Cooper, Jon Lindstrom, Ronald J Lukas, Paul WhiteakerAbstract:Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and Maximum Function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with (125)I-mAb 295 binding and was used to define Function/nAChR. If the α4(+)/(-)β2 sites contribute equally to Function, making identical β2HQT substitutions at either site should produce similar Functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.
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differential α4 β2 agonist binding site contributions to α4β2 nicotinic acetylcholine receptor Function within and between isoforms
Journal of Biological Chemistry, 2016Co-Authors: Linda Lucero, Maegan M Weltzin, Brek J Eaton, John F Cooper, Jon Lindstrom, Ronald J Lukas, Paul WhiteakerAbstract:Abstract Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries, and high vs. low agonist sensitivities, (HS & LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist binding sites in, and between, the isoforms have not been studied. We used a fully-linked subunit concatemeric nAChR approach to express uniform populations of HS- or LS-isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)face E-loop residues to their non-conserved α4 subunit counterparts, or vice-versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. ACh concentration-response curves and Maximum Function were measured using two-electrode voltage-clamp electrophysiology. Surface expression was measured with [125I]mAb 295 binding, and was used to define Function/nAChR. If the α4(+)/(-)β2 sites contribute equally to Function, making identical β2HQT substitutions at either site should produce similar Functional outcomes. Instead, highly-differential outcomes within the HS-isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise-equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation, and that E-loop residues are an important contributor to this neighbor effect.
Florian W R Vondran - One of the best experts on this subject based on the ideXlab platform.
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Use of the liver Maximum Function capacity test (LiMAx) for the management of liver resection in cirrhosis – A case of hypopharyngeal cancer liver metastasis
Elsevier, 2017Co-Authors: S Cammann, J Klempnauer, H Bektas, Felix Oldhafer, Kristina Ringe, Wolf Ramackers, K Timrott, Moritz Kleine, Frank Lehner, Florian W R VondranAbstract:Introduction: The presence of liver cirrhosis goes along with a higher chance for the need of liver resection. As established laboratory parameters often underestimate the degree of cirrhosis this is associated with an increased risk for postoperative liver failure due to the preoperatively impaired liver Function. Known liver Function tests are unlikely to be performed in daily use because of high cost or expenditure of time. Liver Maximum Function capacity test (LiMAx) provides a novel tool for measurement of liver Function and references for the safety of liver resection. Presentation of case: A 63-year old patient presented at our hospital with a large, solitary liver metastasis from hypopharyngeal cancer in segments VII/VIII with infiltration of the diaphragm. Liver resection was unsuccessful in a peripheral hospital 10 months before due to considerable macroscopic liver cirrhosis (CHILD B). Upon presentation conventional laboratory parameters revealed sufficient liver Function. LiMAx was performed and showed regular liver Function (354 μg/kg/h; at norm >315 μg/kg/h). Consequently, atypical liver resection (R0) was performed resulting in a postoperative LiMAx value of 281 μg/h/kg (>150 μg/kg/h). The patient was discharged from hospital 37 days after surgery without any signs of postoperative liver failure. Conclusion: The LiMAx-test enables determination of liver Function at a so far unavailable level (metabolism via cytochrome P450 1A2) and hence might provide crucial additional diagnostic information to allow for safe liver resection even in cirrhotic patients
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use of the liver Maximum Function capacity test limax for the management of liver resection in cirrhosis a case of hypopharyngeal cancer liver metastasis
International Journal of Surgery Case Reports, 2017Co-Authors: S Cammann, J Klempnauer, H Bektas, Felix Oldhafer, Kristina Ringe, Wolf Ramackers, K Timrott, Moritz Kleine, Frank Lehner, Florian W R VondranAbstract:Abstract Introduction The presence of liver cirrhosis goes along with a higher chance for the need of liver resection. As established laboratory parameters often underestimate the degree of cirrhosis this is associated with an increased risk for postoperative liver failure due to the preoperatively impaired liver Function. Known liver Function tests are unlikely to be performed in daily use because of high cost or expenditure of time. Liver Maximum Function capacity test (LiMAx) provides a novel tool for measurement of liver Function and references for the safety of liver resection. Presentation of case A 63-year old patient presented at our hospital with a large, solitary liver metastasis from hypopharyngeal cancer in segments VII/VIII with infiltration of the diaphragm. Liver resection was unsuccessful in a peripheral hospital 10 months before due to considerable macroscopic liver cirrhosis (CHILD B). Upon presentation conventional laboratory parameters revealed sufficient liver Function. LiMAx was performed and showed regular liver Function (354 μg/kg/h; at norm >315 μg/kg/h). Consequently, atypical liver resection (R0) was performed resulting in a postoperative LiMAx value of 281 μg/h/kg (>150 μg/kg/h). The patient was discharged from hospital 37 days after surgery without any signs of postoperative liver failure. Conclusion The LiMAx-test enables determination of liver Function at a so far unavailable level (metabolism via cytochrome P450 1A2) and hence might provide crucial additional diagnostic information to allow for safe liver resection even in cirrhotic patients.
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monitoring of liver Function in a 73 year old patient undergoing associating liver partition and portal vein ligation for staged hepatectomy case report applying the novel liver Maximum Function capacity test
Patient Safety in Surgery, 2016Co-Authors: Felix Oldhafer, J Klempnauer, H Bektas, S Cammann, Kristina Ringe, Wolf Ramackers, K Timrott, Moritz Kleine, Mark D Jager, Florian W R VondranAbstract:Background The two-stage liver resection combining in situ liver transection with portal vein ligation, also referred to as ALPPS (Associating Liver Partition and Portal vein ligation for Staged hepatectomy), has been described as a promising method to increase the resectability of liver tumors. However, one of the most important issues regarding the safety of this procedure is the optimal timing of the second stage at the point of sufficient hypertrophy of the future liver remnant. The recently developed liver Maximum Function capacity test (LiMAx) can be applied to monitor the liver Function postoperatively and hence could be a useful tool for decision-making regarding the timing of the second stage of ALPPS.
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application of the liver Maximum Function capacity test in acute liver failure a helpful tool for decision making in liver transplantation
Case reports in transplantation, 2016Co-Authors: Florian W R Vondran, Carsten Schumacher, Kai Johanning, Bjorn Hartleben, Wolfgang Knitsch, Olaf Wiesner, Elmar Jaeckel, Michael P Manns, J Klempnauer, H BektasAbstract:Background. Despite aggressive intensive medical management acute liver failure (ALF) may require high-urgency liver transplantation (LTx). Available prognostic scores do not apply for all patients; reliable tools to identify individuals in need of LTx are highly required. The liver Maximum Function capacity test (LiMAx) might represent an appropriate option. Referring to a case of ALF after Amanita phalloides-intoxication the potential of the LiMAx-test in this setting is discussed. Presentation of Case. LiMAx was performed in a 27-year-old patient prior to and after high-urgency LTx. In accordance with clinical appearance of hepatic encephalopathy, coagulopathy, and acute kidney failure, the LiMAx-test constituted a fulminant course of ALF with hardly any detectable metabolic activity. Following LTx with a marginal donor organ (95% hepatosteatosis), uptake of liver Function was demonstrated by postoperative increase of the LiMAx-value. The patient was discharged from hospital on postoperative day 26. Discussion. ALF often is associated with a critical state of the patient that requires almost immediate decision-making regarding further therapy. Application of a noninvasive liver Function test might help to determine the prognosis of ALF and support decision-making for or against LTx as well as acceptance of a critical donor organ in case of a critically ill patient.
Maegan M Weltzin - One of the best experts on this subject based on the ideXlab platform.
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differential α4 β2 agonist binding site contributions to α4β2 nicotinic acetylcholine receptor Function within and between isoforms
Journal of Biological Chemistry, 2016Co-Authors: Linda Lucero, Maegan M Weltzin, Brek J Eaton, John F Cooper, Jon Lindstrom, Ronald J Lukas, Paul WhiteakerAbstract:Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and Maximum Function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with (125)I-mAb 295 binding and was used to define Function/nAChR. If the α4(+)/(-)β2 sites contribute equally to Function, making identical β2HQT substitutions at either site should produce similar Functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.
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differential α4 β2 agonist binding site contributions to α4β2 nicotinic acetylcholine receptor Function within and between isoforms
Journal of Biological Chemistry, 2016Co-Authors: Linda Lucero, Maegan M Weltzin, Brek J Eaton, John F Cooper, Jon Lindstrom, Ronald J Lukas, Paul WhiteakerAbstract:Abstract Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries, and high vs. low agonist sensitivities, (HS & LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist binding sites in, and between, the isoforms have not been studied. We used a fully-linked subunit concatemeric nAChR approach to express uniform populations of HS- or LS-isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)face E-loop residues to their non-conserved α4 subunit counterparts, or vice-versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. ACh concentration-response curves and Maximum Function were measured using two-electrode voltage-clamp electrophysiology. Surface expression was measured with [125I]mAb 295 binding, and was used to define Function/nAChR. If the α4(+)/(-)β2 sites contribute equally to Function, making identical β2HQT substitutions at either site should produce similar Functional outcomes. Instead, highly-differential outcomes within the HS-isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise-equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation, and that E-loop residues are an important contributor to this neighbor effect.
John F Cooper - One of the best experts on this subject based on the ideXlab platform.
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differential α4 β2 agonist binding site contributions to α4β2 nicotinic acetylcholine receptor Function within and between isoforms
Journal of Biological Chemistry, 2016Co-Authors: Linda Lucero, Maegan M Weltzin, Brek J Eaton, John F Cooper, Jon Lindstrom, Ronald J Lukas, Paul WhiteakerAbstract:Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist-binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)-face E-loop residues to their non-conserved α4 subunit counterparts or vice versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and Maximum Function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with (125)I-mAb 295 binding and was used to define Function/nAChR. If the α4(+)/(-)β2 sites contribute equally to Function, making identical β2HQT substitutions at either site should produce similar Functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.
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differential α4 β2 agonist binding site contributions to α4β2 nicotinic acetylcholine receptor Function within and between isoforms
Journal of Biological Chemistry, 2016Co-Authors: Linda Lucero, Maegan M Weltzin, Brek J Eaton, John F Cooper, Jon Lindstrom, Ronald J Lukas, Paul WhiteakerAbstract:Abstract Two α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) isoforms exist with (α4)2(β2)3 and (α4)3(β2)2 subunit stoichiometries, and high vs. low agonist sensitivities, (HS & LS), respectively. Both isoforms contain a pair of α4(+)/(-)β2 agonist binding sites. The LS isoform also contains a unique α4(+)/(-)α4 site with lower agonist affinity than the α4(+)/(-)β2 sites. However, the relative roles of the conserved α4(+)/(-)β2 agonist binding sites in, and between, the isoforms have not been studied. We used a fully-linked subunit concatemeric nAChR approach to express uniform populations of HS- or LS-isoform α4β2*-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of β2 subunit (-)face E-loop residues to their non-conserved α4 subunit counterparts, or vice-versa (β2HQT and α4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. ACh concentration-response curves and Maximum Function were measured using two-electrode voltage-clamp electrophysiology. Surface expression was measured with [125I]mAb 295 binding, and was used to define Function/nAChR. If the α4(+)/(-)β2 sites contribute equally to Function, making identical β2HQT substitutions at either site should produce similar Functional outcomes. Instead, highly-differential outcomes within the HS-isoform, and between the two isoforms, were observed. In contrast, α4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise-equivalent α4(+)/(-)β2 agonist sites modifies their contributions to nAChR activation, and that E-loop residues are an important contributor to this neighbor effect.