- Email: [email protected]
The Roles of Selected Pore Residues in Pharmacological Inhibition of the hERG Potassium Channel by a Minimally Structured Blocker
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Tuesday, February 14, 2017
However, a larger construct containing a putative nucleolar signalling sequence (K1147-K1151) labelled the nucleoplasm and the nucleolus, where it colocalizes with fibrillarin. Western blotting of fractionated nuclei confirmed these results. Thus the TRPM7 kinase appears to take different forms, possibly cell specific, allowing it to localize to specific nuclear locations. We surmise that TRPM7 and its cleavage products distribute between the plasma membrane and nucleus in proliferating hepatoma cells. Nucleolar kinase activity may be involved in nucleolar functions such as ribosome synthesis. 2018-Pos Board B338 TRPV1 Contributes to Acrolein-Induced Toxicity Yeranddy A. Alpizar1, Brett Boonen1, Maarten Gees1, Pieter Uvin2, Thomas Voets1, Dirk De Ridder2, Wouter Everaerts2, Karel Talavera1. 1 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium, 2Department of Development and Regeneration, KU Leuven, Leuven, Belgium. Acrolein is a toxic and highly reactive unsaturated aldehyde, often found in cigarette smoke and vehicle exhaust gases. Likewise, acrolein derived from cyclophosphamide-treated patients constitutes the major culprit of bladder irritation during chemotherapy in cancer patient. Although, initially, its toxicity and inflammatory properties have been related to the activation of the transient receptor potential A1 (TRPA1) in nociceptive neurons, recent evidences suggests that other receptor may also play a role in acrolein-induced toxicity. Here we show that, acrolein induces chemical irritation and nocifensive response in the absence of TRPA1. Ratiometric calcium measurements and patch-clamp demonstrate that acrolein activates TRPV1 but, unlike TRPA1 that desensitizes immediately after activation, acrolein-induced activation of TRPV1 is prolonged in time. Furthermore, we identify the N-terminal amino acid residue C157 as key for acrolein-induced TRPV1 activation. Taken together, our results reveal a mechanism underlying the major role of TRPV1 as mediator for the acrolein-induced toxicity, unveiling TRPV1 as a potential therapeutic target in a wide spectrum of noxious conditions, from exposure to smoke to cancer treatment.
Ion Channels, Pharmacology, and Disease I 2019-Pos Board B339 State- and Use-Dependent Binding of KCNQ Channel Openers Caroline K. Wang, Alice W. Wang, Harley Kurata. University of Alberta, Edmonton, AB, Canada. Retigabine is the first approved anticonvulsant that acts via activation of KCNQ2-5 voltage-gated potassium channels, caused by a hyperpolarizing shift in the voltage-dependence of activation. An important unexplored feature of retigabine and its derivatives is their state- and use-dependent properties. Drugs that exhibit use-dependence may have stronger effects with more frequent channel stimulation, enabling selective targeting of hyperexcitable cells. We aimed to generate a detailed understanding of the mechanism of action of various KCNQ channel openers by testing their state- and use-dependent effects on KCNQ2 channels. We assessed drug binding to pre-open states by applying drugs at different holding potentials, and also subjected cells to frequent repetitive stimulation to assess use-dependent drug interactions. WT KCNQ2 channels exhibit marked state- and use- dependent binding to the retigabine analog ICA069673. This drug exclusively binds to activated channel states, with more pronounced effects observed with more frequent channel stimulation. We have also demonstrated that the KCNQ2[A181P] mutant significantly diminishes the gating shift and state-dependent effects of ICA069673. Retigabine exhibits less pronounced gating effects than ICA069673, along with less pronounced state-dependence highlighted by its ability to bind to KCNQ2 at negative holding potentials. We used fast solution exchange to assess kinetics of drug interactions. At positive voltages, ICA069673 binds similarly to both WT KCNQ2 and A181P mutant channels. Upon repolarization to negative voltages however, ICA069673 dramatically decelerates WT KCNQ2 closure, but has much weaker effects on KCNQ2 [A181P]. These data suggest that the active conformation of KCNQ2 [A181P] channels is less effectively stabilized by ICA069673. These findings highlight the dramatic state-dependence of certain KCNQ channel openers and begin to reveal the underlying molecular determinants of these effects. 2020-Pos Board B340 Functional Annotation of KCNQ1 Variants of Unknown Significance using Automated Electrophysiology Carlos G. Vanoye1, Reshma R. Desai1, Katarina L. Fabre1, Jens Meiler2, Charles R. Sanders3, Alfred L. George1. 1 Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA, 2Chemistry, Vanderbilt University, Nashville, TN, USA, 3 Biochemistry, Vanderbilt University, Nashville, TN, USA.
Channelopathies result from ion channel dysfunction caused by mutation in either pore-forming subunits or regulatory proteins. The widespread use of genetic and genomic testing has led to an explosive growth in the number of newly discovered ion channel variants associated with human diseases and in reference populations. For example, in congenital long QT syndrome (LQTS), hundreds of variants have been discovered in KCNQ1 and KCNE1, which encode the pore-forming subunit and accessory protein required to generate the slow delayed cardiac rectifier current (IKs). Functional annotation experiments (e.g., patch-clamp recording) have become the gold standard in assessing the likely pathogenicity of ion channel variants, but the extreme time- and labor-intensity of this approach is insufficient to tackle hundreds, if not thousands, of known variants. To overcome this challenge, we have implemented a work flow that combined high efficiency electroporation to achieve transient co-expression of ion channel subunits in cultured cells with automated planar patch clamp recording performed in a 384-well format. We demonstrated the success of this approach by determining the functional properties of 51 KCNQ1 variants co-expressed with KCNE1 in CHO cells. Channel subunits were expressed from plasmid vectors encoding either EGFP (KCNQ1) or DsRed (KCNE1) enabling quantification of transfection efficiency by flow cytometry. Approximately 2/3 of cells were typically co-transfected with both subunits. Automated patch-clamp recording was performed before and after application of the specific IKs blocker HMR1556 (20 mM), enabling offline subtraction of non-specific currents. Typically, ~80-90% of wells exhibited cell capture, high seal resistance (>0.5Gohms) and low series resistance. Semi-automated data handling routines allowed for rapid analysis of current density, voltage-dependence of activation and gating kinetics. Our results indicate a successful implementation of a robust workflow that enables rapid functional annotation of human ion channel variants. 2021-Pos Board B341 The Effects of Extracellular Protons on the hERG Potassium Channel Stacey L. Wilson, Neil V. Marrion, Jules C. Hancox. Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom. The a-subunit of channels mediating the cardiac rapid delayed rectifier current (IKr) is encoded by the human Ether-a`-go-go-Related Gene (hERG). Macroscopic hERG current (IhERG) amplitude is reduced and deactivation kinetics are accelerated with extracellular acidosis. We have investigated the single channel basis for the effects of acidic external pH (pHe) on the isoforms of IhERG expressed in myocytes (hERG1a and 1b). Patch clamp recordings were made at room temperature with the extracellular superfusate (wholecell) or pipette solution (cell attached) acidified to pH 6.3 compared with control (pH 7.4). A decrease in pHe to 6.3 caused acceleration in deactivation and a reduction in maximal whole-cell conductance of ~34% for IhERG1a (n=8 cells) and of ~36% for IhERG1b (n=5 cells). Single channel recordings were made with isotonic potassium (140 mM) bathing the cells and in the electrode. Channel amplitude and open state kinetics were measured at a series of repolarisation voltages following a depolarising command to þ40mV. Slope conductance values derived from amplitude current-voltage relationships between 120 and 40mV were 12.350.2pS for pH 7.4 (n=10 cells) and 9.350.1pS for pH 6.3 (n=9 cells) (P<0.01, two-tailed t-test) for hERG1a. The corresponding values for hERG1b were 11.450.2pS for pH 7.4 (n=6 cells) and 7.650.4pS for pH 6.3 (n=5 cells) (P<0.0001; two-tailed t-test). Open-time kinetics at 120mV for hERG1a were reduced from 8.4951.0ms in control (n=8 cells) to 4.250.4ms in pHe 6.3 (n=7 cells) (P<0.05; two-tailed t-test). The hERG1b open state kinetics were 5.750.6ms in pHe 7.4 (n=6 cells) and reduced to 3.150.7ms in pHe 6.3 (n=5 cells) (P<0.05; two-tailed t-test). Thus, it can be concluded that a reduction in the single channel conductance and acceleration of opentimes contribute to the attenuation of macroscopic IhERG when exposed to acidic pHe. 2022-Pos Board B342 The Roles of Selected Pore Residues in Pharmacological Inhibition of the hERG Potassium Channel by a Minimally Structured Blocker Matthew V. Helliwell1, Jules C. Hancox2, Yi H. Zhang2, Christopher E. Dempsey1. 1 Department of Biochemistry, University of Bristol, Bristol, United Kingdom, 2Department of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom. hERG Kþ channels are sensitive to pharmacological inhibition by structurally diverse drugs. This can lead to acquired long QT syndrome. We have characterised the interactions with hERG of a recently designed ‘‘minimally structured’’ high affinity hERG channel inhibitor, ‘‘Cavalli-2’’. Experiments
Tuesday, February 14, 2017 were performed on HEK-293 cells expressing wild-type or mutant hERG channels. Whole-cell patch clamp measurements of IhERG were made at 37 C and data are presented as mean 5 SEM, nR5. Commercially synthesized Cavalli-2 suppressed hERG tail currents (IhERG) with a half-maximal inhibitory concentration (IC50) of 35.6 5 1.0 nM. Aromatic residues on the S6 helix are known to be structural determinants for high affinity hERG channel block. Mutation of Y652 residues to alanine markedly reduced the affinity of Cavalli-2 for hERG (100 nM Cavalli-2 caused 61.5 5 5.0% block of WT IhERG but only 23.0 5 4.1% of Y652A IhERG). The pore helical residues T623 and S624 have been reported to make important interactions with polar aromatic para-substituents of drugs. Despite the absence of polar para-substituents in the minimal hERG blocker, T623A IhERG (which required measuring inward tail currents in high [Kþ] at 120 mV), was blocked 47.5 5 4.2% by 100 nM Cavalli-2 compared to 73.8 5 7.1% of inward WT IhERG and S624A IhERG was blocked by 29.1 5 3.8% (compared to 61.5 5 5.0% WT IhERG). Combined with computational modelling of interactions of Cavalli-2 with the hERG pore, these results support binding modes in which S624 stabilises the protonated amino group of Cavalli-2 near the internal K binding site and T623 may promote favourable pore geometries for drug binding, possibly by stabilising optimal conformations of Y652. 2023-Pos Board B343 Lamprey CFTR, an Evolutionary Ancestor of Human CFTR, Exhibits Numerous Biophysical Dissimilarities from Human CFTR Guiying Cui1, Brandon B. Stauffer1, Barry R. Imhoff1, Amit Gaggar2, Nael A. McCarty1. 1 Pediatrics, Emory University, Atlanta, GA, USA, 2Division of Pulmonary, Allergy & Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham, Brimingham, AL, USA. Lampreys, an extant representative of the jawless vertebrates, diverged from jawed vertebrates approximately 550 million years ago and possess a unique CFTR ortholog. Lamprey CFTR (Lp-CFTR) shares 46% sequence identity and 65% sequence similarity with human CFTR (hCFTR). The biophysical consequences of this dissimilarity remain unknown so we investigated the channel behavior and pharmacology of Lp-CFTR expressed in Xenopus oocytes. Like hCFTR, Lp-CFTR channel activity was PKA-dependent with whole cell currents stimulated by forskolin and IBMX, and excised macropatch currents activated by intracellular exposure to MgATP and PKA. Two blockers of hCFTR, NPPB and glibenclamide, blocked Lp-CFTR activity in a manner comparable to hCFTR. Surprisingly, GlyH-101 failed to block Lp-CFTR at Vm = 60 mV and CFTRinh172 exhibited modest inhibition of Lp-CFTR in both the whole cell (TEVC) and inside-out macropatch configuration. The sole clinical hCFTR potentiator VX-770 (KalydecoTM) mildly potentiated hCFTR in the presence of MgATP þ PKA, recorded with the inside-out macropatch technique. However, Lp-CFTR was significantly inhibited by VX-770 under the same experimental conditions. Furthermore, Lp-CFTR exhibited significant inward rectification, the most among all the CFTR species tested thus far, when it was recorded in symmetrical 150 mM Cl- in inside-out macropatches. The single channel behavior of Lp-CFTR was also unique. Whereas open hCFTR channels occupy the full open state (f) over 98% of the time, Lp-CFTR randomly occupies the f, the subconductance 1 (s1), or subconductance 2 (s2) states. In summary, LpCFTR has unique pharmacological and biophysical characteristics compared to hCFTR. The differences may provide a tool for identifying the binding sites and working mechanism of VX-770 and may shed light on understanding the evolution of CFTR. (NIH R01-DK 056481). 2024-Pos Board B344 Small Molecule Induced Changes in the Trafficking of the Cystic Fibrosis Conductance Regulator Zhihui Zhang, David Heidary, Chris Richards. Chemistry, University of Kentucky, Lexington, KY, USA. Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Deletion of Phe508 (F508), the most prevalent mutation in CFTR, results in the failure of CFTR trafficking to plasma membrane. My work focused on understanding changes in the trafficking of F508 CFTR to the plasma membrane due to the correction of the small molecule VX809, low temperature (30 C ) and second site mutations (I539T). Real time total internal reflection fluorescence (TIRF) images were taken to identify whether correctors increased surface expression through increased trafficking or membrane turnover. The pH sensitive fluorescence protein, super ecliptic phluorin, was tagged to CFTR to quantify plasma membrane expression. The second site mutation I539T and low temperature alone increase F508 CFTR
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trafficking to approximately 10% of wild type level, while the combination of the small molecule VX809 with either the second site mutation or low temperature increased trafficking to nearly 40% of wild type level. The result indicates that the small molecule VX809 works synergistically with either the low temperature or the second site mutation. No statistical difference is seen between the cargo load in each trafficking vesicle of wild type and I539T/ VX809 corrected F508 CFTR. Overall, the results suggest both low temperatures and second site mutations lead to the stabilization of a pool of mutant CFTR in the ER. VX809 acts on this stabilized pool increasing trafficking to the plasma membrane. 2025-Pos Board B345 Characterization of CFTR Activators and Inhibitors by the use of a Planar Patch Clamp System Andrea Br€uggemann1, Soren Friis2, Tim Strassmeier3, Markus Rapedius1, Tom Goetze1, Ilka Rinke1, Claudia Haarmann1, Nina Brinkwirth1, Atsushi Ohtsuki4, Takayuki Oka4, Michael George1, Niels Fertig1. 1 Nanion Technologies, Munich, Germany, 2Nanion Technologies, Copenhagen, Denmark, 3Nanion Technologies, NJ, USA, 4Nanion Technologies, Tokio, Japan. Cystic fibrosis is caused by malfunction of the chloride channel Cystic Fibrosis Transmembrane Regulator (CFTR). CFTR is expressed in the apical membrane of epithelial cells where it is involved in the regulation of fluid transport across the epithelium. A large number of mutations in the protein are known to cause CFTR to become dysfunctional and only a few pharmaceutical compounds have been developed to treat the disease by restoring the chloride conductance of the channel (1). CFTR is activated by cAMP dependent phosphorylation and is gated by ATP. Activation is typically achieved using forskolin that activates adenylate cyclase which then leads to phosphorylation of the channel via protein kinase A (PKA). Screening for CFTR activators is usually done in the absence of forskolin, but in the presence of low concentrations of cAMP. Studies in animal models support the development of CFTR inhibitors for antisecretory therapy of enterotoxin-mediated diarrheas and polycystic kidney disease. For this purpose an alternative approach can be used. In the presence of internal fluoride CFTR is activated as to the same degree as after forskolin activation. We here present data from Nanion’s SyncroPatch 384PE where we activate the channel with either forskolin under fluoride free conditions or by internal solution exchange where the fluoride is washed into the intracellular solution. Our results show that the activation of CFTR by either forskolin or intracellular F- is sensitive to glibenclamide and CFTRInh-172 in a dose and/or voltage dependent manner. Taken together, these experiments show a stable and cost optimized approach to study the pharmacology of CFTR at high throughput that might empowers new ways in the drug discovery on CFTR. 2026-Pos Board B346 Discovery of NAV1.7 Modulators from Marketed Drugs using High throuthput Automated Electrophysiological System Ionworks Barracuda Xueqin Chen, Sui Fang, Qiang Ding, Zhaobing Gao. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China. The voltage-gated sodium channel Nav1.7 is a major contributor to pain signaling in humans and a subject of extensive research programs as an important target for developing anti-pain drugs. Finding good drug leads de novo from large chemical libraries, real or virtual, is not an easy task. Especially for an ion channel drug target, using indirect high-throughput screening such as fluorescence-based assay is often plagued by a significant level of false positives, low hit rates and many leads that are toxic or exhibit poor bioavailability. Exploiting the secondary activity of marketed drugs, on the other hand, may help in generating drug leads that can be optimized for the observed side-effect target, while maintaining acceptable bioavailability and toxicity profiles. Here, we described an efficient high throughput electrophysiology assay to discover hits to the Nav1.7 target from safe marketed drugs. We screened a marketed drugs library(US Drug Collection 1280 drugs) using IonWorks Barracuda(IWB) platform(population patch clamp mode, PPC), the second generation of IonWorks instruments. Two different voltage protocols were used to compare compounds inhibition between resting and inactivation state of the channel. We tested throughput, reproducibility and uniformity of the Nav1.7/IWB assay and demonstrated that the assay had met standard industry acceptance criteria as Z0 factor, signal window and inter-plate reproducibility of IC50 values for positive controls. Results showed that 41 drugs has an inhibition percentage of more than 80% on Nav1.7 channel, some of them are anesthetic such as Tetracaine, Benoxinate and Diperodon.
Tuesday, February 14, 2017
However, a larger construct containing a putative nucleolar signalling sequence (K1147-K1151) labelled the nucleoplasm and the nucleolus, where it colocalizes with fibrillarin. Western blotting of fractionated nuclei confirmed these results. Thus the TRPM7 kinase appears to take different forms, possibly cell specific, allowing it to localize to specific nuclear locations. We surmise that TRPM7 and its cleavage products distribute between the plasma membrane and nucleus in proliferating hepatoma cells. Nucleolar kinase activity may be involved in nucleolar functions such as ribosome synthesis. 2018-Pos Board B338 TRPV1 Contributes to Acrolein-Induced Toxicity Yeranddy A. Alpizar1, Brett Boonen1, Maarten Gees1, Pieter Uvin2, Thomas Voets1, Dirk De Ridder2, Wouter Everaerts2, Karel Talavera1. 1 Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium, 2Department of Development and Regeneration, KU Leuven, Leuven, Belgium. Acrolein is a toxic and highly reactive unsaturated aldehyde, often found in cigarette smoke and vehicle exhaust gases. Likewise, acrolein derived from cyclophosphamide-treated patients constitutes the major culprit of bladder irritation during chemotherapy in cancer patient. Although, initially, its toxicity and inflammatory properties have been related to the activation of the transient receptor potential A1 (TRPA1) in nociceptive neurons, recent evidences suggests that other receptor may also play a role in acrolein-induced toxicity. Here we show that, acrolein induces chemical irritation and nocifensive response in the absence of TRPA1. Ratiometric calcium measurements and patch-clamp demonstrate that acrolein activates TRPV1 but, unlike TRPA1 that desensitizes immediately after activation, acrolein-induced activation of TRPV1 is prolonged in time. Furthermore, we identify the N-terminal amino acid residue C157 as key for acrolein-induced TRPV1 activation. Taken together, our results reveal a mechanism underlying the major role of TRPV1 as mediator for the acrolein-induced toxicity, unveiling TRPV1 as a potential therapeutic target in a wide spectrum of noxious conditions, from exposure to smoke to cancer treatment.
Ion Channels, Pharmacology, and Disease I 2019-Pos Board B339 State- and Use-Dependent Binding of KCNQ Channel Openers Caroline K. Wang, Alice W. Wang, Harley Kurata. University of Alberta, Edmonton, AB, Canada. Retigabine is the first approved anticonvulsant that acts via activation of KCNQ2-5 voltage-gated potassium channels, caused by a hyperpolarizing shift in the voltage-dependence of activation. An important unexplored feature of retigabine and its derivatives is their state- and use-dependent properties. Drugs that exhibit use-dependence may have stronger effects with more frequent channel stimulation, enabling selective targeting of hyperexcitable cells. We aimed to generate a detailed understanding of the mechanism of action of various KCNQ channel openers by testing their state- and use-dependent effects on KCNQ2 channels. We assessed drug binding to pre-open states by applying drugs at different holding potentials, and also subjected cells to frequent repetitive stimulation to assess use-dependent drug interactions. WT KCNQ2 channels exhibit marked state- and use- dependent binding to the retigabine analog ICA069673. This drug exclusively binds to activated channel states, with more pronounced effects observed with more frequent channel stimulation. We have also demonstrated that the KCNQ2[A181P] mutant significantly diminishes the gating shift and state-dependent effects of ICA069673. Retigabine exhibits less pronounced gating effects than ICA069673, along with less pronounced state-dependence highlighted by its ability to bind to KCNQ2 at negative holding potentials. We used fast solution exchange to assess kinetics of drug interactions. At positive voltages, ICA069673 binds similarly to both WT KCNQ2 and A181P mutant channels. Upon repolarization to negative voltages however, ICA069673 dramatically decelerates WT KCNQ2 closure, but has much weaker effects on KCNQ2 [A181P]. These data suggest that the active conformation of KCNQ2 [A181P] channels is less effectively stabilized by ICA069673. These findings highlight the dramatic state-dependence of certain KCNQ channel openers and begin to reveal the underlying molecular determinants of these effects. 2020-Pos Board B340 Functional Annotation of KCNQ1 Variants of Unknown Significance using Automated Electrophysiology Carlos G. Vanoye1, Reshma R. Desai1, Katarina L. Fabre1, Jens Meiler2, Charles R. Sanders3, Alfred L. George1. 1 Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA, 2Chemistry, Vanderbilt University, Nashville, TN, USA, 3 Biochemistry, Vanderbilt University, Nashville, TN, USA.
Channelopathies result from ion channel dysfunction caused by mutation in either pore-forming subunits or regulatory proteins. The widespread use of genetic and genomic testing has led to an explosive growth in the number of newly discovered ion channel variants associated with human diseases and in reference populations. For example, in congenital long QT syndrome (LQTS), hundreds of variants have been discovered in KCNQ1 and KCNE1, which encode the pore-forming subunit and accessory protein required to generate the slow delayed cardiac rectifier current (IKs). Functional annotation experiments (e.g., patch-clamp recording) have become the gold standard in assessing the likely pathogenicity of ion channel variants, but the extreme time- and labor-intensity of this approach is insufficient to tackle hundreds, if not thousands, of known variants. To overcome this challenge, we have implemented a work flow that combined high efficiency electroporation to achieve transient co-expression of ion channel subunits in cultured cells with automated planar patch clamp recording performed in a 384-well format. We demonstrated the success of this approach by determining the functional properties of 51 KCNQ1 variants co-expressed with KCNE1 in CHO cells. Channel subunits were expressed from plasmid vectors encoding either EGFP (KCNQ1) or DsRed (KCNE1) enabling quantification of transfection efficiency by flow cytometry. Approximately 2/3 of cells were typically co-transfected with both subunits. Automated patch-clamp recording was performed before and after application of the specific IKs blocker HMR1556 (20 mM), enabling offline subtraction of non-specific currents. Typically, ~80-90% of wells exhibited cell capture, high seal resistance (>0.5Gohms) and low series resistance. Semi-automated data handling routines allowed for rapid analysis of current density, voltage-dependence of activation and gating kinetics. Our results indicate a successful implementation of a robust workflow that enables rapid functional annotation of human ion channel variants. 2021-Pos Board B341 The Effects of Extracellular Protons on the hERG Potassium Channel Stacey L. Wilson, Neil V. Marrion, Jules C. Hancox. Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom. The a-subunit of channels mediating the cardiac rapid delayed rectifier current (IKr) is encoded by the human Ether-a`-go-go-Related Gene (hERG). Macroscopic hERG current (IhERG) amplitude is reduced and deactivation kinetics are accelerated with extracellular acidosis. We have investigated the single channel basis for the effects of acidic external pH (pHe) on the isoforms of IhERG expressed in myocytes (hERG1a and 1b). Patch clamp recordings were made at room temperature with the extracellular superfusate (wholecell) or pipette solution (cell attached) acidified to pH 6.3 compared with control (pH 7.4). A decrease in pHe to 6.3 caused acceleration in deactivation and a reduction in maximal whole-cell conductance of ~34% for IhERG1a (n=8 cells) and of ~36% for IhERG1b (n=5 cells). Single channel recordings were made with isotonic potassium (140 mM) bathing the cells and in the electrode. Channel amplitude and open state kinetics were measured at a series of repolarisation voltages following a depolarising command to þ40mV. Slope conductance values derived from amplitude current-voltage relationships between 120 and 40mV were 12.350.2pS for pH 7.4 (n=10 cells) and 9.350.1pS for pH 6.3 (n=9 cells) (P<0.01, two-tailed t-test) for hERG1a. The corresponding values for hERG1b were 11.450.2pS for pH 7.4 (n=6 cells) and 7.650.4pS for pH 6.3 (n=5 cells) (P<0.0001; two-tailed t-test). Open-time kinetics at 120mV for hERG1a were reduced from 8.4951.0ms in control (n=8 cells) to 4.250.4ms in pHe 6.3 (n=7 cells) (P<0.05; two-tailed t-test). The hERG1b open state kinetics were 5.750.6ms in pHe 7.4 (n=6 cells) and reduced to 3.150.7ms in pHe 6.3 (n=5 cells) (P<0.05; two-tailed t-test). Thus, it can be concluded that a reduction in the single channel conductance and acceleration of opentimes contribute to the attenuation of macroscopic IhERG when exposed to acidic pHe. 2022-Pos Board B342 The Roles of Selected Pore Residues in Pharmacological Inhibition of the hERG Potassium Channel by a Minimally Structured Blocker Matthew V. Helliwell1, Jules C. Hancox2, Yi H. Zhang2, Christopher E. Dempsey1. 1 Department of Biochemistry, University of Bristol, Bristol, United Kingdom, 2Department of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom. hERG Kþ channels are sensitive to pharmacological inhibition by structurally diverse drugs. This can lead to acquired long QT syndrome. We have characterised the interactions with hERG of a recently designed ‘‘minimally structured’’ high affinity hERG channel inhibitor, ‘‘Cavalli-2’’. Experiments
Tuesday, February 14, 2017 were performed on HEK-293 cells expressing wild-type or mutant hERG channels. Whole-cell patch clamp measurements of IhERG were made at 37 C and data are presented as mean 5 SEM, nR5. Commercially synthesized Cavalli-2 suppressed hERG tail currents (IhERG) with a half-maximal inhibitory concentration (IC50) of 35.6 5 1.0 nM. Aromatic residues on the S6 helix are known to be structural determinants for high affinity hERG channel block. Mutation of Y652 residues to alanine markedly reduced the affinity of Cavalli-2 for hERG (100 nM Cavalli-2 caused 61.5 5 5.0% block of WT IhERG but only 23.0 5 4.1% of Y652A IhERG). The pore helical residues T623 and S624 have been reported to make important interactions with polar aromatic para-substituents of drugs. Despite the absence of polar para-substituents in the minimal hERG blocker, T623A IhERG (which required measuring inward tail currents in high [Kþ] at 120 mV), was blocked 47.5 5 4.2% by 100 nM Cavalli-2 compared to 73.8 5 7.1% of inward WT IhERG and S624A IhERG was blocked by 29.1 5 3.8% (compared to 61.5 5 5.0% WT IhERG). Combined with computational modelling of interactions of Cavalli-2 with the hERG pore, these results support binding modes in which S624 stabilises the protonated amino group of Cavalli-2 near the internal K binding site and T623 may promote favourable pore geometries for drug binding, possibly by stabilising optimal conformations of Y652. 2023-Pos Board B343 Lamprey CFTR, an Evolutionary Ancestor of Human CFTR, Exhibits Numerous Biophysical Dissimilarities from Human CFTR Guiying Cui1, Brandon B. Stauffer1, Barry R. Imhoff1, Amit Gaggar2, Nael A. McCarty1. 1 Pediatrics, Emory University, Atlanta, GA, USA, 2Division of Pulmonary, Allergy & Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham, Brimingham, AL, USA. Lampreys, an extant representative of the jawless vertebrates, diverged from jawed vertebrates approximately 550 million years ago and possess a unique CFTR ortholog. Lamprey CFTR (Lp-CFTR) shares 46% sequence identity and 65% sequence similarity with human CFTR (hCFTR). The biophysical consequences of this dissimilarity remain unknown so we investigated the channel behavior and pharmacology of Lp-CFTR expressed in Xenopus oocytes. Like hCFTR, Lp-CFTR channel activity was PKA-dependent with whole cell currents stimulated by forskolin and IBMX, and excised macropatch currents activated by intracellular exposure to MgATP and PKA. Two blockers of hCFTR, NPPB and glibenclamide, blocked Lp-CFTR activity in a manner comparable to hCFTR. Surprisingly, GlyH-101 failed to block Lp-CFTR at Vm = 60 mV and CFTRinh172 exhibited modest inhibition of Lp-CFTR in both the whole cell (TEVC) and inside-out macropatch configuration. The sole clinical hCFTR potentiator VX-770 (KalydecoTM) mildly potentiated hCFTR in the presence of MgATP þ PKA, recorded with the inside-out macropatch technique. However, Lp-CFTR was significantly inhibited by VX-770 under the same experimental conditions. Furthermore, Lp-CFTR exhibited significant inward rectification, the most among all the CFTR species tested thus far, when it was recorded in symmetrical 150 mM Cl- in inside-out macropatches. The single channel behavior of Lp-CFTR was also unique. Whereas open hCFTR channels occupy the full open state (f) over 98% of the time, Lp-CFTR randomly occupies the f, the subconductance 1 (s1), or subconductance 2 (s2) states. In summary, LpCFTR has unique pharmacological and biophysical characteristics compared to hCFTR. The differences may provide a tool for identifying the binding sites and working mechanism of VX-770 and may shed light on understanding the evolution of CFTR. (NIH R01-DK 056481). 2024-Pos Board B344 Small Molecule Induced Changes in the Trafficking of the Cystic Fibrosis Conductance Regulator Zhihui Zhang, David Heidary, Chris Richards. Chemistry, University of Kentucky, Lexington, KY, USA. Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Deletion of Phe508 (F508), the most prevalent mutation in CFTR, results in the failure of CFTR trafficking to plasma membrane. My work focused on understanding changes in the trafficking of F508 CFTR to the plasma membrane due to the correction of the small molecule VX809, low temperature (30 C ) and second site mutations (I539T). Real time total internal reflection fluorescence (TIRF) images were taken to identify whether correctors increased surface expression through increased trafficking or membrane turnover. The pH sensitive fluorescence protein, super ecliptic phluorin, was tagged to CFTR to quantify plasma membrane expression. The second site mutation I539T and low temperature alone increase F508 CFTR
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trafficking to approximately 10% of wild type level, while the combination of the small molecule VX809 with either the second site mutation or low temperature increased trafficking to nearly 40% of wild type level. The result indicates that the small molecule VX809 works synergistically with either the low temperature or the second site mutation. No statistical difference is seen between the cargo load in each trafficking vesicle of wild type and I539T/ VX809 corrected F508 CFTR. Overall, the results suggest both low temperatures and second site mutations lead to the stabilization of a pool of mutant CFTR in the ER. VX809 acts on this stabilized pool increasing trafficking to the plasma membrane. 2025-Pos Board B345 Characterization of CFTR Activators and Inhibitors by the use of a Planar Patch Clamp System Andrea Br€uggemann1, Soren Friis2, Tim Strassmeier3, Markus Rapedius1, Tom Goetze1, Ilka Rinke1, Claudia Haarmann1, Nina Brinkwirth1, Atsushi Ohtsuki4, Takayuki Oka4, Michael George1, Niels Fertig1. 1 Nanion Technologies, Munich, Germany, 2Nanion Technologies, Copenhagen, Denmark, 3Nanion Technologies, NJ, USA, 4Nanion Technologies, Tokio, Japan. Cystic fibrosis is caused by malfunction of the chloride channel Cystic Fibrosis Transmembrane Regulator (CFTR). CFTR is expressed in the apical membrane of epithelial cells where it is involved in the regulation of fluid transport across the epithelium. A large number of mutations in the protein are known to cause CFTR to become dysfunctional and only a few pharmaceutical compounds have been developed to treat the disease by restoring the chloride conductance of the channel (1). CFTR is activated by cAMP dependent phosphorylation and is gated by ATP. Activation is typically achieved using forskolin that activates adenylate cyclase which then leads to phosphorylation of the channel via protein kinase A (PKA). Screening for CFTR activators is usually done in the absence of forskolin, but in the presence of low concentrations of cAMP. Studies in animal models support the development of CFTR inhibitors for antisecretory therapy of enterotoxin-mediated diarrheas and polycystic kidney disease. For this purpose an alternative approach can be used. In the presence of internal fluoride CFTR is activated as to the same degree as after forskolin activation. We here present data from Nanion’s SyncroPatch 384PE where we activate the channel with either forskolin under fluoride free conditions or by internal solution exchange where the fluoride is washed into the intracellular solution. Our results show that the activation of CFTR by either forskolin or intracellular F- is sensitive to glibenclamide and CFTRInh-172 in a dose and/or voltage dependent manner. Taken together, these experiments show a stable and cost optimized approach to study the pharmacology of CFTR at high throughput that might empowers new ways in the drug discovery on CFTR. 2026-Pos Board B346 Discovery of NAV1.7 Modulators from Marketed Drugs using High throuthput Automated Electrophysiological System Ionworks Barracuda Xueqin Chen, Sui Fang, Qiang Ding, Zhaobing Gao. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China. The voltage-gated sodium channel Nav1.7 is a major contributor to pain signaling in humans and a subject of extensive research programs as an important target for developing anti-pain drugs. Finding good drug leads de novo from large chemical libraries, real or virtual, is not an easy task. Especially for an ion channel drug target, using indirect high-throughput screening such as fluorescence-based assay is often plagued by a significant level of false positives, low hit rates and many leads that are toxic or exhibit poor bioavailability. Exploiting the secondary activity of marketed drugs, on the other hand, may help in generating drug leads that can be optimized for the observed side-effect target, while maintaining acceptable bioavailability and toxicity profiles. Here, we described an efficient high throughput electrophysiology assay to discover hits to the Nav1.7 target from safe marketed drugs. We screened a marketed drugs library(US Drug Collection 1280 drugs) using IonWorks Barracuda(IWB) platform(population patch clamp mode, PPC), the second generation of IonWorks instruments. Two different voltage protocols were used to compare compounds inhibition between resting and inactivation state of the channel. We tested throughput, reproducibility and uniformity of the Nav1.7/IWB assay and demonstrated that the assay had met standard industry acceptance criteria as Z0 factor, signal window and inter-plate reproducibility of IC50 values for positive controls. Results showed that 41 drugs has an inhibition percentage of more than 80% on Nav1.7 channel, some of them are anesthetic such as Tetracaine, Benoxinate and Diperodon.