Feb 14, 2017 - Tania Chernov-Rogan1, Tianbo Li1, Henry Verschoof2, Kuldip Khakh2, ... 1Pharmacology, University of California Davis, Davis, CA, USA,.
Tuesday, February 14, 2017 1631-Plat A Novel Membrane Potential Assay to Identify Nav1.7-Selective Blockers Tania Chernov-Rogan1, Tianbo Li1, Henry Verschoof2, Kuldip Khakh2, Steven Jones1, Steve McKerrall1, David H. Hackos1, Dan Sutherlin1, Charles J. Cohen2, Jun Chen1. 1 Genentech, South San Francisco, CA, USA, 2Xenon, Burnaby, BC, Canada. The voltage-gated sodium channel Nav1.7 is one of the most validated pain targets, as gain-of-function mutations cause excessive pain, whereas loss-offunction mutations produce insensitivity to pain. Targeting Nav1.7 holds great promise in treating pain, yet presents a tantalizing challenge, as few Nav1.7 selective chemical scaffolds exist. So far millions of compounds have been screened by using fluorescence membrane potential (MP) assays, but very few selective hits have been identified. Here we show that the conventional MP approach, which uses Nav1.7 WT channel and the activator veratridine, is intrinsically flawed: it is biased toward non-selective pore blockers which compete with veratridine, and fails to detect highly potent, selective compounds such as the aryl sulfonamide PF-771. By using a mutant channel and an alternative activator, we developed a novel method that not only robustly detects known Nav1.7 blockers, but also decreases the number of non-selective pore blocker hits. We conducted a high-throughput screen using this methodology and identified novel Nav1.7-selective blockers. 1632-Plat High Diastolic Sodium Influx in Heart Failure has Drug-Sensitivities Like Late Sodium Current, but Produces Inward Current at Diastolic Potentials Zhandi Liao1, Kenneth S. Ginsburg1, Daniel C. Bartos1, Yanyan Jiang1, Sanda Despa2, Donald M. Bers1. 1 Pharmacology, University of California Davis, Davis, CA, USA, 2 Pharmacology and Nutritional Sciences, University of Kentucky, Lexington, KY, USA. Heart failure (HF) affects over 6 million people, enhances arrhythmogenesis and is associated with elevated intracellular [Na] ([Na]i), which impacts Ca handling via Na/Ca exchange. HF myocytes exhibit increased late Na current (INaL), typically insufficient to explain the rise in [Na]i. However, in HF there is also an increase in diastolic Na influx (INaDiast) that is tetrodotoxin (TTX) sensitive. INaL is known to be activated by CaMKII and blocked by the selective INaL inhibitors ranolazine and GS967. To test whether the elevated INaDiast in HF has the same sensitivities to CaMKII and INaL inhibitors, we studied freshly isolated ventricular myocytes from HF and control rabbits. We measured [Na]i and unidirectional Na-influx rate upon acute Na/K-ATPase blockade. Ranolazine (10 mM) inhibited INaDiast in HF myocytes (by 58.5%), consistent with INaDiast in HF being mediated via an INaL-like current. GS967, tested in control myocytes, also inhibited INaDiast. Application of CaMKII blocker, KN-93 (1 mM) reduced INaDiast in HF, but not control myocytes. We also measured INaDiast under voltage clamp, as shifts in holding current (Ihold) at diastolic Vm upon acute addition of INaL blockers. In control myocytes TTX induced 7.5pA IHold, but in HF myocytes the TTX-sensitive IHold was 25.4 pA at 80mV. This agrees with INaDiast measured using Na dyes. INaDiast could be via a window INa. However, the V1/2 of the INa steady state activation and inactivation curves were shifted in HF by 3.8mV and 7.2mV, respectively, and intermediate INa inactivation was also enhanced in HF. Both of these effects make window INa current unlikely to mediate INaDiast. INaL measured in APclamp was increased by ~50% in HF. We suggest that INaDiast in HF has drug sensitivities like INaL during the AP, but produces inward current at diastolic potentials that can explain the rise in [Na]i. 1633-Plat Voltage Gated Proton Channel Expression and Function in Breast Cancer Cells Dan Bare1, Vladimir V. Cherny1, Abde M. Abukhdeir2, Thomas E. DeCoursey1, Deri Morgan1. 1 Molecular Biophysics, Rush University Medical Center, Chicago, IL, USA, 2 Medicine and Pharmacology, Rush University Medical Center, Chicago, IL, USA. Immunohistochemical evidence for voltage gated proton channels (hHV1) was recently reported in breast cancer cells, but not healthy breast tissue. Higher levels of hHV1 expression were correlated with advanced stages of the disease, worse prognosis, and decreased survival [Wang et al, 2012, J.Biol.Chem. 287:13877]. However, its function and mechanism of action have yet to be elucidated. Here, we examine a number of breast cancer cell lines for expression of hHV1 and explored the consequences of hHV1 genetic knockout. Using patch-clamp, we demonstrated that MDA-MB-231 cells have hHV1 currents. The activation threshold, gating kinetics, selectivity, and pH dependence of the channel were similar to those in other human cells. We screened across
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all subtypes of breast cancer (Luminal A, Luminal B, Her2, and ‘‘triple negative’’) using 12 different cancer cell lines for the presence of the hHV1 protein in membrane fractions by western blotting. Significant hHV1 expression was found only in 3 of the 6 triple-negative cell lines tested. Immunostaining of MDA-MB-231 cells demonstrated that the channel resided in the plasma membrane, with little or no expression in the cytosol. CRIPSR/ Cas9-mediated deletion of the hHV1 gene did not detectably change the recovery of MDA-MB-231 cells from an acid load. In contrast, 100 mM Zn2þ profoundly slowed recovery. Na/Hþ exchange appeared to be the dominant mechanism in cytosolic pH recovery. Nevertheless, deletion of hHV1 resulted in multiple changes in protein expression, assessed by reverse phase protein analysis, including increased COX2 and lowered CD171 expression in MDA-MB-231 cells in three independent clones, as compared to wild-type and Cas9-expressing controls. These data suggest a mechanism outside of pH regulation for hHV1 in breast cancer cells and caution when using Zn2þ as a pharmacological agent to test for hHV1 function. Supported by NIH-GM102336, Bears Care, and Gavers Community Cancer Fund. 1634-Plat BK Channel Activation in Chronic Vasodilation by Thiazide-Like Diuretics: Role of the Beta-1 Auxiliary Subunit Pedro Martı´n, Agustı´n Asuaje, Valentina Pastore, Vero´nica Milesi. Instituto de estudios Inmunolo´gicos y Fisiopatolo´gicos (IIFP UNLP-CONICET), La Plata, Argentina. Thiazide-like diuretics are still recommended as first-line antihypertensive therapy, based on their chronic vasodilatory effects. Previous studies suggest activation of the large conductance voltage- and Ca2þ- dependent Kþ channel (BK channel) expressed in vascular smooth muscle cells (SMCs), as responsible for this vasodilator effect, but electrophysiological evidence supporting this is lacking. BK can be accompanied by accessory b-subunits, which confer specific pharmacological characteristics to the channel. The b1 subunit is mainly expressed in SMCs. We measured the effect of hydrochlorothiazide (HCTZ) on BK channel activity using the patch-clamp technique in SMCs from human umbilical artery (HUASMCs) and in HEK293 T expressing the BK channel (with and without the beta-1 subunit). In HUASMCs, HCTZ (10 mM) caused significant activation of the BK current in the whole-cell configuration (528 5 215 to 1379 5 132 pA at þ40mV, n: 4, p 0.05), suggesting an indirect activation mechanism. In HEK cells expressing the BK channel (with and without b1-subunit), HCTZ only activates BK channel in the presence of the b1-subunit. This activation was concentration-dependent with an EC50 of 28 mM (pD2=4.546 5 0.211, n: 5-8). Membrane potential did not influence the concentration relationship on HCTZ-induced BK channel activation. Consistently, HCTZ did not change the BK channel activity when it was evaluated in HEK cells expressing the b1-subunit in the inside-out configuration, where cell integrity is lost. These results suggest that the vasodilatory effects of HCTZ could be due to an indirect activation of the BK channel depending on the beta-1 subunit expression. 1635-Plat Differential KD Channels Expression in ‘‘Classically’’ and ‘‘Alternatively’’ Activated Microglia Hai M. Nguyen1, Eva M. Gro¨ssinger1, Makoto Horiuchi2, Kyle W. Davis2, Lee-Way Jin2, Izumi Maezawa2, Heike Wulff1. 1 Pharmacology, University of California, Davis, Davis, CA, USA, 2 Pathology and Laboratory Medicine, M.I.N.D. Institute, University of California, Davis, Sacramento, CA, USA. As CNS resident immune cells, microglia are highly active and can assume different phenotypes in response to microenvironmental signals. Lipopolysaccharide (LPS) and interferon-g (IFN-g) promote differentiation into classically activated M1-like microglia, which produce high levels of pro-inflammatory cytokines and nitric oxide and are thought to contribute to neurological damage in ischemic stroke and Alzheimer’s disease. IL-4 in contrast induces a phenotype associated with anti-inflammatory effects and tissue repair. We here investigated whether these microglia subsets vary in their Kþ channel expression by differentiating neonatal mouse microglia into M(LPS) and M(IL-4) microglia and studying their Kþ channel expression by whole-cell patch-clamp, quantitative PCR and immunohistochemistry. We identified three major types of Kþ channels based on their biophysical and pharmacological
