Abstract

We have previously identified Ser293 in transmembrane segment 5 as a determinant for selective K_Ca2.1 channel activation by GW542573X (4-(2-methoxyphenylcarbamoyloxymethyl)-piperidine-1-carboxylic acid tert-butyl ester). Now we show that Ser293 mediates both activation and inhibition of K_Ca2.1: CM-TPMF (N-{7-[1-(4-chloro-2-methylphenoxy)ethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-N′-methoxy-formamidine) and B-TPMF (N-{7-[1-(4-tert-butyl-phenoxy)ethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-N′-methoxy-formamidine), two newly identified and structurally related [1,2,4]triazolo[1,5-a]pyrimidines, act either as activators or as inhibitors of the human K_Ca2.1 channel. Whereas (−)-CM-TPMF activates K_Ca2.1 with an EC₅₀ value of 24 nM, (−)-B-TPMF inhibits the channel with an IC₅₀ value of 31 nM. In contrast, their (+)-enantiomers are 40 to 100 times less active. Both (−)-CM-TPMF and (−)-B-TPMF are subtype-selective, with 10- to 20-fold discrimination toward other K_Ca2 channels and the K_Ca3 channel. Coapplication experiments reveal competitive-like functional interactions between the effects of (−)-CM-TPMF and (−)-B-TPMF. Despite belonging to a different chemical class than GW542573X, the K_Ca2.1 selectivity of (−)-CM-TPMF and (−)-B-TPMF depend critically on Ser293 as revealed by loss- and gain-of-function mutations. We conclude that compounds occupying the TPMF site may either positively or negatively influence the gating process depending on their substitution patterns. It is noteworthy that (−)-CM-TPMF is 10 times more potent on K_Ca2.1 than NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime), an unselective but hitherto the most potent K_Ca3/K_Ca2 channel activator. (−)-B-TPMF is the first small-molecule inhibitor with significant selectivity among the K_Ca2 channel subtypes. In contrast to peptide blockers such as apamin and scyllatoxin, which preferentially affect K_Ca2.2, (−)-B-TPMF exhibits K_Ca2.1 selectivity. These high-affinity compounds, which exert opposite effects on K_Ca2.1 gating, may help define physiological or pathophysiological roles of this channel.