Abstract
A new small molecule, 4-(2-methoxy-phenylcarbamoyloxymethyl)-piperidine-1-carboxylic acid tert-butyl ester (GW542573X), is presented as an activator of small-conductance Ca(2+)-activated K(+) (SK, K(Ca)2) channels and distinguished from previously published positive modulators of SK channels, such as 1-ethyl-2-benzimidazolinone (1-EBIO) and cyclohexyl-[2-(3,5-dimethylpyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA), in several aspects. GW542573X is the first SK1-selective compound described: an EC(50) value of 8.2 +/- 0.8 microM (n = 6, [Ca(2+)](i) = 200 nM) was obtained from inside-out patches excised from hSK1-expressing HEK293 cells. Whole-cell experiments showed that hSK2 and hSK3 channels were more than 10 times, and hIK channels even more than 100 times, less sensitive to GW542573X. The Ca(2+)-response curve of hSK1 was left-shifted from an EC(50)(Ca(2+)) value of 410 +/- 20 nM (n = 9) to 240 +/- 10 nM (n = 5) in the presence of 10 microM GW542573X. In addition to this positive modulation, GW542573X activated SK1 in the absence of Ca(2+) and furthermore induced a 15% increase in the maximal current at saturating Ca(2+). Thus, GW542573X also acts as a genuine opener of the hSK1 channels, a mechanism of action (MOA) not previously obtained with SK channels. The differential potency on hSK1 and hSK3 enabled a chimera approach to elucidate site(s) important for this new MOA and selectivity property. A single amino acid (Ser293) located in S5 of hSK1 was essential, and substituting the corresponding Leu476 in hSK3 with serine conferred hSK1-like potency (EC(50) = 9.3 +/- 1.4 microM, n = 5). GW542573X may activate SK channels via interaction with "deep-pore" gating structures at the inner pore vestibule or the selectivity filter in contrast to 1-EBIO and CyPPA that exert positive modulation via the intracellular calmodulin binding domain.