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062P
University
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Identification of (E)-2-(4,6-difluoro-1-indanylidene)acetamide (GW275919X) as a novel opener of human recombinant IK and SK Ca2+-activated K+ channels J.E. Cryan, T.J. Dale, 1M.X. Chen, 2G. Robertson & D.J. Trezise Ion Channel Biology Group, Systems Research, 1Gene Expression & Protein Biochemistry, and 2High Throughput Chemistry, GlaxoSmithKline, Medicines Research Centre, Stevenage, Hertfordshire, SG1 2NY, United Kingdom. |
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Ca2+-activated K+ channels (SK-1 to 3, IK) are widely expressed in excitable and non-excitable cells and serve to link increases in [Ca2+]i with membrane hyperpolarisation (see Jensen et al., 2001 for review). Delineation of the (patho)physiological roles of these channels would be aided by the identification of small molecule openers, but thus far, only a few, generally weakly active agents are known (e.g. 1-ethyl-2-benzimidazolinone (1-EBIO) and chlorzoxazone). Here we describe the in vitro properties of (E)-2-(4,6-difluoro-1-indanylidene) acetamide (GW275919X), a more potent novel, structurally distinct opener of human recombinant Ca2+-activated K+ channels.
The construction of cell lines stably expressing either IK or SK channels has been previously described (HEK-hIK, CHO-hSK-1, CHO-hSK-2 and CHO-hSK-3; Dale et al., 2002). Whole-cell patch-clamp electrophysiology recordings were made (Dale et al., 2002) under symmetrical K+ solutions (external solution (mM): KCl (144), MgCl2 (1), CaCl2 (2), glucose (5) and HEPES (10), pH 7.4; internal solution (mM): K-gluconate (144), MgCl2 (1.15), EGTA (5), CaCl2 (2.06) and HEPES (10), pH 7.25, both 290-310 mOsm). This yielded a free (unchelated) [Ca2+]i of 100nM (Brooks & Storey, 1992). Voltage ramps (-100mV to +70mV, 1s) were applied every 5s. Application of GW275919X (0.3-100µM) evoked reversible, concentration-related increases in K+ current in hIK-CHO cells(mean 7.8nA±2.0nA at 100µM). The geometric mean EC50 value for GW275919X was 7µM [95% confidence limits 4-13µM] (n=4), some 20-fold more potent than 1-EBIO (136mM [80-231] (n=7)). GW275919X (100µM) did not evoke currents in wild type CHO cells. The specific IK channel blocker, charybdotoxin (100nM) markedly attenuated activation by GW275919X (30µM; 74±3% inhibition, n=3; P<0.05). GW275919X also activated hSK-1, hSK-2 and hSK-3 with mean EC50 values of 91µM [59-139], 211µM [142-314] and 170µM [144-202] (n=3-5), respectively. The activation of hIK by GW275919X was strongly Ca2+-dependent - fixing free [Ca2+]i at 30nM, 100nM and 300nM by modifying the internal CaCl2/EGTA ratio yielded EC50 values of 67µM (n=5), 7µM (n=4) and 2mM (n=4), respectively. In current clamp experiments (144mM [K+]i, 4.7mM [K+]o) GW275919X (100mM) hyperpolarised the membrane potential from -21±1mV to -85±1mV, a value close to the theoretical K+ equilibrium potential (n=4).
We conclude that GW275919X is a novel K+ channel opener that is 10-30 fold selective for IK channels over SK channels, and 20-fold more potent than 1-EBIO. This agent should be a useful tool for further characterising the physiological role of IK and SK channels.
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Jensen, B.S. et al. (2001). Curr. Drug Targets, 2,
401-22.