Evidence for a common pharmacological interaction site on K(Ca)2 channels providing both selective activation and selective inhibition of the human K(Ca)2.1 subtype

TY - JOUR

T1 - Evidence for a common pharmacological interaction site on K(Ca)2 channels providing both selective activation and selective inhibition of the human K(Ca)2.1 subtype

AU - Hougaard, Charlotte

AU - Hammami, Sofia

AU - Eriksen, Birgitte L

AU - Sørensen, Ulrik S

AU - Jensen, Marianne L

AU - Strøbæk, Dorte

AU - Christophersen, Palle

AU - Bomholtz, Sofia Hammami

PY - 2012/2

Y1 - 2012/2

N2 - We have previously identified Ser293 in transmembrane segment 5 as a determinant for selective K(Ca)2.1 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(Ca)2.1: 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(Ca)2.1 channel. Whereas (-)-CM-TPMF activates K(Ca)2.1 with an EC(50) value of 24 nM, (-)-B-TPMF inhibits the channel with an IC(50) 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(Ca)2 channels and the K(Ca)3 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(Ca)2.1 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(Ca)2.1 than NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime), an unselective but hitherto the most potent K(Ca)3/K(Ca)2 channel activator. (-)-B-TPMF is the first small-molecule inhibitor with significant selectivity among the K(Ca)2 channel subtypes. In contrast to peptide blockers such as apamin and scyllatoxin, which preferentially affect K(Ca)2.2, (-)-B-TPMF exhibits K(Ca)2.1 selectivity. These high-affinity compounds, which exert opposite effects on K(Ca)2.1 gating, may help define physiological or pathophysiological roles of this channel.

AB - We have previously identified Ser293 in transmembrane segment 5 as a determinant for selective K(Ca)2.1 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(Ca)2.1: 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(Ca)2.1 channel. Whereas (-)-CM-TPMF activates K(Ca)2.1 with an EC(50) value of 24 nM, (-)-B-TPMF inhibits the channel with an IC(50) 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(Ca)2 channels and the K(Ca)3 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(Ca)2.1 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(Ca)2.1 than NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime), an unselective but hitherto the most potent K(Ca)3/K(Ca)2 channel activator. (-)-B-TPMF is the first small-molecule inhibitor with significant selectivity among the K(Ca)2 channel subtypes. In contrast to peptide blockers such as apamin and scyllatoxin, which preferentially affect K(Ca)2.2, (-)-B-TPMF exhibits K(Ca)2.1 selectivity. These high-affinity compounds, which exert opposite effects on K(Ca)2.1 gating, may help define physiological or pathophysiological roles of this channel.

KW - Amino Acid Substitution

KW - Binding Sites

KW - Humans

KW - Inhibitory Concentration 50

KW - Ion Channel Gating

KW - Small-Conductance Calcium-Activated Potassium Channels

KW - Stereoisomerism

KW - Structure-Activity Relationship

U2 - 10.1124/mol.111.074252

DO - 10.1124/mol.111.074252

M3 - Journal article

C2 - 22046005

SN - 0026-895X

VL - 81

SP - 210

EP - 219

JO - Molecular Pharmacology

JF - Molecular Pharmacology

IS - 2

ER -