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

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 Ca²⁺-activated K⁺ (SK, K_Ca2) 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.