Abstract
We have previously identified Ser293 in transmembrane segment 5 as a determinant for selective KCa2.1 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 KCa2.1: 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 KCa2.1 channel. Whereas (−)-CM-TPMF activates KCa2.1 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 KCa2 channels and the KCa3 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 KCa2.1 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 KCa2.1 than NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime), an unselective but hitherto the most potent KCa3/KCa2 channel activator. (−)-B-TPMF is the first small-molecule inhibitor with significant selectivity among the KCa2 channel subtypes. In contrast to peptide blockers such as apamin and scyllatoxin, which preferentially affect KCa2.2, (−)-B-TPMF exhibits KCa2.1 selectivity. These high-affinity compounds, which exert opposite effects on KCa2.1 gating, may help define physiological or pathophysiological roles of this channel.
Footnotes
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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ABBREVIATIONS:
- KCNN
- small and intermediate conductance Ca2+-activated K+ channel
- CaM
- calmodulin
- CaMBD
- CaM binding domain
- CNS
- central nervous system
- 1-EBIO
- 1-ethyl-2-benzimidazolinone
- UCL1684
- 6,10-diaza-3(1,3),8(1,4)-dibenzena-1,5(1,4)-diquinolinacyclodecaphane
- DCEBIO
- 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one
- NS4591
- 4,5-dichloro-1,3-diethyl-2,3-dihydro-1H-1,3-benzodiazol-2-one
- SKA-31
- naphtho[1,2-d]thiazol-2-ylamine
- NS8593
- (R)-N-(benzimidazol-2-yl)-tetrahydro-1-naphtylamine
- NS309
- 6,7-dichloro-1H-indole-2,3-dione 3-oxime
- GW542573X
- 4-(2-methoxyphenylcarbamoyloxymethyl)-piperidine-1-carboxylic acid tert-butyl ester
- B-TPMF
- (N-{7-[1-(4-tert-butyl-phenoxy)ethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-N′-methoxy-formamidine
- CM-TPMF
- N-{7-[1-(4-chloro-2-methylphenoxy)ethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-N′-methoxy-formamidine
- 1-EBIO
- 1-ethyl-2-benzimidazolinone
- HEK
- human embryonic kidney
- I-V
- current-voltage
- Bay-k-8644
- S-(−)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester
- KCa2
- small conductance Ca2+-activated K+ channel (SK channel)
- KCa3.1
- intermediate conductance Ca2+-activated K+ channel (IK channel).
- Received June 22, 2011.
- Accepted November 1, 2011.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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