scholarly journals Sulfonylurea Receptor Transmembrane Domain Zero Mutations that Disrupt Full Length and Minimal ATP-Sensitive Potassium Channel Properties

2010 ◽  
Vol 98 (3) ◽  
pp. 699a
Author(s):  
Emily B. Pratt ◽  
Paul Tewson ◽  
Show-Ling Shyng
Keyword(s):  
Potassium Channel ◽  
Transmembrane Domain ◽  
Full Length ◽  
Sulfonylurea Receptor ◽  
Channel Properties

scholarly journals N-terminal transmembrane domain of SUR1 controls gating of Kir6.2 by modulating channel sensitivity to PIP2

2011 ◽  
Vol 137 (3) ◽  
pp. 299-314 ◽  
Author(s):  
Emily B. Pratt ◽  
Paul Tewson ◽  
Cathrin E. Bruederle ◽  
William R. Skach ◽  
Show-Ling Shyng
Keyword(s):  
Katp Channel ◽  
Transmembrane Domain ◽  
Katp Channels ◽  
Full Length ◽  
Regulatory Subunit ◽  
Functional Coupling ◽  
Sulfonylurea Receptor ◽  
Open Probability ◽  
Protein Levels ◽  
Channel Inactivation

Functional integrity of pancreatic adenosine triphosphate (ATP)-sensitive potassium (KATP) channels depends on the interactions between the pore-forming potassium channel subunit Kir6.2 and the regulatory subunit sulfonylurea receptor 1 (SUR1). Previous studies have shown that the N-terminal transmembrane domain of SUR1 (TMD0) interacts with Kir6.2 and is sufficient to confer high intrinsic open probability (Po) and bursting patterns of activity observed in full-length KATP channels. However, the nature of TMD0–Kir6.2 interactions that underlie gating modulation is not well understood. Using two previously described disease-causing mutations in TMD0 (R74W and E128K), we performed amino acid substitutions to study the structural roles of these residues in KATP channel function in the context of full-length SUR1 as well as TMD0. Our results revealed that although R74W and E128K in full-length SUR1 both decrease surface channel expression and reduce channel sensitivity to ATP inhibition, they arrive there via distinct mechanisms. Mutation of R74 uniformly reduced TMD0 protein levels, suggesting that R74 is necessary for stability of TMD0. In contrast, E128 mutations retained TMD0 protein levels but reduced functional coupling between TMD0 and Kir6.2 in mini-KATP channels formed by TMD0 and Kir6.2. Importantly, E128K full-length channels, despite having a greatly reduced Po, exhibit little response to phosphatidylinositol 4,5-bisphosphate (PIP2) stimulation. This is reminiscent of Kir6.2 channel behavior in the absence of SUR1 and suggests that TMD0 controls Kir6.2 gating by modulating Kir6.2 interactions with PIP2. Further supporting this notion, the E128W mutation in full-length channels resulted in channel inactivation that was prevented or reversed by exogenous PIP2. These results identify a critical determinant in TMD0 that controls Kir6.2 gating by controlling channel sensitivity to PIP2. Moreover, they uncover a novel mechanism of KATP channel inactivation involving aberrant functional coupling between SUR1 and Kir6.2.

Molecular Mechanisms of the Inhibitory Effects of Bupivacaine, Levobupivacaine, and Ropivacaine on Sarcolemmal Adenosine Triphosphate–sensitive Potassium Channels in the Cardiovascular System

2004 ◽  
Vol 101 (2) ◽  
pp. 390-398 ◽  
Author(s):  
Takashi Kawano ◽  
Shuzo Oshita ◽  
Akira Takahashi ◽  
Yasuo Tsutsumi ◽  
Yoshinobu Tomiyama ◽  
...  
Keyword(s):  
Cardiovascular System ◽  
Adenosine Triphosphate ◽  
Local Anesthetics ◽  
Molecular Mechanisms ◽  
Transmembrane Domain ◽  
Katp Channels ◽  
Inhibitory Effect ◽  
Amino Acid Residues ◽  
Sulfonylurea Receptor ◽  
Inhibitory Effects

Background Sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channels in the cardiovascular system may be involved in bupivacaine-induced cardiovascular toxicity. The authors investigated the effects of local anesthetics on the activity of reconstituted KATP channels encoded by inwardly rectifying potassium channel (Kir6.0) and sulfonylurea receptor (SUR) subunits. Methods The authors used an inside-out patch clamp configuration to investigate the effects of bupivacaine, levobupivacaine, and ropivacaine on the activity of reconstituted KATP channels expressed in COS-7 cells and containing wild-type, mutant, or chimeric SURs. Results Bupivacaine inhibited the activities of cardiac KATP channels (IC50 = 52 microm) stereoselectively (levobupivacaine, IC50 = 168 microm; ropivacaine, IC50 = 249 microm). Local anesthetics also inhibited the activities of channels formed by the truncated isoform of Kir6.2 (Kir6.2 delta C36) stereoselectively. Mutations in the cytosolic end of the second transmembrane domain of Kir6.2 markedly decreased both the local anesthetics' affinity and stereoselectivity. The local anesthetics blocked cardiac KATP channels with approximately eightfold higher potency than vascular KATP channels; the potency depended on the SUR subtype. The 42 amino acid residues at the C-terminal tail of SUR2A, but not SUR1 or SUR2B, enhanced the inhibitory effect of bupivacaine on the Kir6.0 subunit. Conclusions Inhibitory effects of local anesthetics on KATP channels in the cardiovascular system are (1) stereoselective: bupivacaine was more potent than levobupivacaine and ropivacaine; and (2) tissue specific: local anesthetics blocked cardiac KATP channels more potently than vascular KATP channels, via the intracellular pore mouth of the Kir6.0 subunit and the 42 amino acids at the C-terminal tail of the SUR2A subunit, respectively.

A Transmembrane Domain of the Sulfonylurea Receptor Mediates Activation of ATP-Sensitive K+ Channels by K+Channel Openers

1999 ◽  
Vol 56 (2) ◽  
pp. 308-315 ◽  
Author(s):  
Nathalie D’hahan ◽  
Hélène Jacquet ◽  
Christophe Moreau ◽  
Patrice Catty ◽  
Michel Vivaudou
Keyword(s):  
Transmembrane Domain ◽  
K Channel ◽  
Sulfonylurea Receptor ◽  
K Channels

scholarly journals Similarities and differences between native HIV-1 envelope glycoprotein trimers and stabilized soluble trimer mimetics

2018 ◽  
Author(s):  
Alba Torrents de la Peña ◽  
Kimmo Rantalainen ◽  
Christopher A. Cottrell ◽  
Joel D. Allen ◽  
Marit J. van Gils ◽  
...  
Keyword(s):  
Envelope Glycoprotein ◽  
Neutralizing Antibodies ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Full Length ◽  
Broadly Neutralizing Antibodies ◽  
Vaccine Research ◽  
Similarities And Differences ◽  
Membrane Tether ◽  
Hiv 1

AbstractThe HIV-1 envelope glycoprotein (Env) trimer is located on the surface of the virus and is the target of broadly neutralizing antibodies (bNAbs). Recombinant native-like soluble Env trimer mimetics, such as SOSIP trimers, have taken a central role in HIV-1 vaccine research aimed at inducing bNAbs. We therefore performed a direct and thorough comparison of a full-length native Env trimer containing the transmembrane domain and the cytoplasmic tail, with the sequence matched soluble SOSIP trimer, both based on an early Env sequence (AMC011) from an HIV+ individual that developed bNAbs. The structures of the full-length AMC011 trimer bound to either bNAb PGT145 or PGT151 were very similar to the structures of SOSIP trimers. Antigenically, the full-length and SOSIP trimers were comparable, but in contrast to the full-length trimer, the SOSIP trimer did not bind at all to non-neutralizing antibodies, most likely as a consequence of the intrinsic stabilization of the SOSIP trimer. Furthermore, the glycan composition of full-length and SOSIP trimers was similar overall, but the SOSIP trimer possessed slightly less complex and less extensively processed glycans, which may relate to the intrinsic stabilization as well as the absence of the membrane tether. These data provide insights into how to best use and improve membrane-associated full-length and soluble SOSIP HIV-1 Env trimers as immunogens.

Abstract 1515: Regional Effects of Katp Channel Openers Reflect Distinct Atrial and Ventricular Katp Channel Structure in the Murine Heart

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Alexey V Glukhov ◽  
Thomas P Flagg ◽  
Vadim V Fedorov ◽  
Igor R Efimov ◽  
Colin G Nichols
Keyword(s):  
Left Ventricle ◽  
Right Ventricle ◽  
Potassium Channel ◽  
Katp Channel ◽  
Channel Structure ◽  
Receptor Type ◽  
Optical Data ◽  
Sulfonylurea Receptor ◽  
Spatiotemporal Resolution ◽  
Intact Mouse

Classically, cardiac sarcolemmal KATP channels are thought to be composed of Kir6.2 (inward-rectifier potassium channel 6.2, KCNJ11) and SUR2A (sulfonylurea receptor type 2A, ABCC9) subunits. However, the evidence is strong that SUR1 (sulfonylurea receptor type 1, ABCC8) subunits are also expressed in the heart and that they play a significant functional role in the atria. To examine this further, we have estimated the effects of potassium channel-opening drugs diazoxide (specific to SUR1 > SUR2A) and pinacidil (SUR2A > SUR1) in intact hearts from wild type (WT, n=6) and SUR1−/− mice (KO, n=3) that lack SUR1 subunits. Action potential duration (APD) in both atria and ventricles were estimated by optical mapping of posterior surface of Langendorff-perfused hearts using the voltage sensitive dye RH237 and high spatiotemporal resolution CMOS camera (100x100 pixels; 3,000 frames/sec). In WT hearts, diazoxide (300 μM) decreased APD in atria (from 33.6±2.1 ms to 25.5±1.0 ms, p<0.001) and did not change it in ventricles (60.0±8.5 ms vs 61.2±8.3 ms, NS). The absence of SUR1 in KO mice resulted in loss of efficacy of diazoxide in atria (37.5±0.7 ms vs 36.5±0.7 ms, NS). In contrast, pinacidil (300 μM) significantly decreased ventricular APD in both type of mice (from 60.0±8.5 ms to 30.5±4.2 ms in WT, p<0.001; and from 62.0±1.4 ms to 30.5±6.4 ms in KO, p<0.001) and did not change atrial APD in either WT or KO hearts. Glass microelectrode recordings from isolated superfused atria confirmed the optical data. In both WT and KO hearts, the APD in left ventricle was significantly longer and the effect of pinacidil was significantly greater than in right ventricle (APD decreasing by 56.3±4.2% and 62.3±12.0% in left ventricle vs 49.4±3.4% and 50.9±4.1% in right ventricle for WT and KO mice respectively, p<0.05). Similar differences between APDs in right and left atria were not observed. Collectively, these results indicate that in the intact mouse heart, significant differential KATP pharmacology in atria and ventricles results from SUR1 predominance in forming the atrial channel, highlighting an unappreciated heterogeneity of KATP function in the heart.

scholarly journals Comparative gain-of-function effects of the KCNMA1-N999S mutation on human BK channel properties

2020 ◽  
Vol 123 (2) ◽  
pp. 560-570 ◽  
Author(s):  
Hans J. Moldenhauer ◽  
Katia K. Matychak ◽  
Andrea L. Meredith
Keyword(s):  
Action Potential ◽  
Potassium Channel ◽  
Antiepileptic Drug ◽  
Bk Channel ◽  
Gain Of Function ◽  
Double Mutation ◽  
Brain Physiology ◽  
Number Of Patients ◽  
Calcium Activated Potassium Channel ◽  
Channel Properties

KCNMA1, encoding the voltage- and calcium-activated potassium channel, has a pivotal role in brain physiology. Mutations in KCNMA1 are associated with epilepsy and/or dyskinesia (PNKD3). Two KCNMA1 mutations correlated with these phenotypes, D434G and N999S, were previously identified as producing gain-of-function (GOF) effects on BK channel activity. Three new patients have been reported harboring N999S, one carrying a second mutation, R1128W, but the effects of these mutations have not yet been reported under physiological K+ conditions or compared to D434G. In this study, we characterize N999S, the novel N999S/R1128W double mutation, and D434G in a brain BK channel splice variant, comparing the effects on BK current properties under a physiological K+ gradient with action potential voltage commands. N999S, N999S/R1128W, and D434G cDNAs were expressed in HEK293T cells and characterized by patch-clamp electrophysiology. N999S BK currents were shifted to negative potentials, with faster activation and slower deactivation compared with wild type (WT) and D434G. The double mutation N999S/R1128W did not show any additional changes in current properties compared with N999S alone. The antiepileptic drug acetazolamide was assessed for its ability to directly modulate WT and N999S channels. Neither the WT nor N999S channels were sensitive to the antiepileptic drug acetazolamide, but both were sensitive to the inhibitor paxilline. We conclude that N999S is a strong GOF mutation that surpasses the D434G phenotype, without mitigation by R1128W. Acetazolamide has no direct modulatory action on either WT or N999S channels, indicating that its use may not be contraindicated in patients harboring GOF KCNMA1 mutations. NEW & NOTEWORTHY KCNMA1-linked channelopathy is a new neurological disorder characterized by mutations in the BK voltage- and calcium-activated potassium channel. The epilepsy- and dyskinesia-associated gain-of-function mutations N999S and D434G comprise the largest number of patients in the cohort. This study provides the first direct comparison between D434G and N999S BK channel properties as well as a novel double mutation, N999S/R1128W, from another patient, defining the functional effects during an action potential stimulus.

scholarly journals Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

2001 ◽  
Vol 69 (12) ◽  
pp. 7839-7850 ◽  
Author(s):  
Gillian D. Pullinger ◽  
R. Sowdhamini ◽  
Alistair J. Lax
Keyword(s):  
Amino Acids ◽  
Fusion Proteins ◽  
Pasteurella Multocida ◽  
Transmembrane Domain ◽  
Polyclonal Antibodies ◽  
Full Length ◽  
Cell Binding ◽  
Terminal Fragment ◽  
C Terminus ◽  
N Terminus

ABSTRACT The locations of the catalytic and receptor-binding domains of thePasteurella multocida toxin (PMT) were investigated. N- and C-terminal fragments of PMT were cloned and expressed as fusion proteins with affinity tags. Purified fusion proteins were assessed in suitable assays for catalytic activity and cell-binding ability. A C-terminal fragment (amino acids 681 to 1285) was catalytically active. When microinjected into quiescent Swiss 3T3 cells, it induced changes in cell morphology typical of toxin-treated cells and stimulated DNA synthesis. An N-terminal fragment with a His tag at the C terminus (amino acids 1 to 506) competed with full-length toxin for binding to surface receptors and therefore contains the cell-binding domain. The inactive mutant containing a mutation near the C terminus (C1165S) also bound to cells in this assay. Polyclonal antibodies raised to the N-terminal PMT region bound efficiently to full-length native toxin, suggesting that the N terminus is surface located. Antibodies to the C terminus of PMT were microinjected into cells and inhibited the activity of toxin added subsequently to the medium, confirming that the C terminus contains the active site. Analysis of the PMT sequence predicted a putative transmembrane domain with predicted hydrophobic and amphipathic helices near the N terminus over the region of homology to the cytotoxic necrotizing factors. The C-terminal end of PMT was predicted to be a mixed α/β domain, a structure commonly found in catalytic domains. Homology to proteins of known structure and threading calculations supported these assignments.

scholarly journals Antibody responses to rhesus cytomegalovirus glycoprotein B in naturally infected rhesus macaques

2003 ◽  
Vol 84 (12) ◽  
pp. 3371-3379 ◽  
Author(s):  
Yujuan Yue ◽  
Shan Shan Zhou ◽  
Peter A. Barry
Keyword(s):  
Specific Antibody ◽  
Transmembrane Domain ◽  
Solid Phase ◽  
Rhesus Macaques ◽  
Antibody Responses ◽  
Full Length ◽  
Glycoprotein B ◽  
Primate Model ◽  
Specific Antibodies ◽  
Rhesus Cytomegalovirus

Rhesus cytomegalovirus (RhCMV) exhibits strong parallels with human CMV (HCMV) in terms of nucleic and amino acid identities, natural history, and mechanisms of persistence and pathogenesis in its natural host, rhesus macaques (Macaca mulatta). To determine whether this non-human primate model would be useful to assess vaccine strategies for HCMV, host immune responses to RhCMV glycoprotein B (gB) were evaluated in RhCMV-infected monkeys. Total protein extracts were prepared from cells transiently transfected with an expression plasmid for either the full-length gB or a derivative (gBΔ, 1–680 aa) lacking both the transmembrane domain and cytoplasmic tail. Western blot analysis showed identical reactivity of macaque sera with full-length gB and its derivative gBΔ, indicating that the immunodominant epitopes of gB are contained in the extracellular portion of the protein. Using gBΔ extract as a solid phase, a sensitive and specific ELISA was established to characterize gB antibody responses in monkeys acutely and chronically infected with RhCMV. During primary infection (seroconversion), gB-specific antibodies developed concurrently and in parallel with total RhCMV-specific antibodies. However, during chronic infection gB-specific antibody responses were variable. A strong correlation was observed between neutralizing and gB-specific antibody levels in RhCMV-seropositive monkeys. Taken together, the results of this study indicate that, similar to host humoral responses to HCMV gB, anti-gB antibodies are an integral part of humoral immunity to RhCMV infection and probably play an important protective role in limiting the extent of RhCMV infection. Thus, the rhesus macaque model of HCMV infection is relevant for testing gB-based immune therapies.

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