Selective activation of the SK1 subtype of human small-conductance Ca2+-activated K+ channels by 4-(2-methoxyphenylcarbamoyloxymethyl)-piperidine-1-carboxylic acid tert-butyl ester (GW542573X) is dependent on serine 293 in the S5 segment.
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.
Hougaard
Charlotte
C
NeuroSearch A/S, Pederstrupvej 93, DK 2750 Ballerup, Denmark.
Jensen
Marianne L
ML
Dale
Tim J
TJ
Miller
David D
DD
Davies
David J
DJ
Eriksen
Birgitte L
BL
Strøbaek
Dorte
D
Trezise
Derek J
DJ
Christophersen
Palle
P
D019943:Amino Acid Substitution; D002219:Carbamates / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D006801:Humans; D009154:Mutation; D010880:Piperidines / Q000737:chemistry / Q000494:pharmacology*; D012694:Serine / Q000235:genetics; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000819:agonists* / Q000235:genetics*
C543324:4-(2-methoxyphenylcarbamoyloxymethyl)piperidine-1-carboxylic acid tert-butyl ester; D002219:Carbamates; C496897:KCNN2 protein, human; D010880:Piperidines; D051640:Small-Conductance Calcium-Activated Potassium Channels; D012694:Serine