RT Journal Article
SR Electronic
T1 Selective Activation of the SK1 Subtype of Human Small-Conductance Ca<sup>2+</sup>-Activated K<sup>+</sup> Channels by 4-(2-Methoxyphenylcarbamoyloxymethyl)-piperidine-1-carboxylic Acid <em>tert</em>-Butyl Ester (GW542573X) Is Dependent on Serine 293 in the S5 Segment
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 569
OP 578
DO 10.1124/mol.109.056663
VO 76
IS 3
A1 Charlotte Hougaard
A1 Marianne L. Jensen
A1 Tim J. Dale
A1 David D. Miller
A1 David J. Davies
A1 Birgitte L. Eriksen
A1 Dorte Strøbæk
A1 Derek J. Trezise
A1 Palle Christophersen
YR 2009
UL http://molpharm.aspetjournals.org/content/76/3/569.abstract
AB A new small molecule, 4-(2-methoxy-phenylcarbamoyloxymethyl)-piperidine-1-carboxylic acid tert-butyl ester (GW542573X), is presented as an activator of small-conductance Ca2+-activated K+ (SK, KCa2) 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 EC50 value of 8.2 ± 0.8 μM (n = 6, [Ca2+]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 Ca2+-response curve of hSK1 was left-shifted from an EC50(Ca2+) value of 410 ± 20 nM (n = 9) to 240 ± 10 nM (n = 5) in the presence of 10 μM GW542573X. In addition to this positive modulation, GW542573X activated SK1 in the absence of Ca2+ and furthermore induced a 15% increase in the maximal current at saturating Ca2+. 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 (EC50 = 9.3 ± 1.4 μM, 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.