Abstract
Acting as a negative gating modulator, (R)-N-(benzimidazol-2-yl)-1,2,3,4- tetrahydro-1-naphthylamine (NS8593) shifts the apparent Ca2+- dependence of the small-conductance Ca2+-activated K+ channels KCa2.1-2.3 to higher Ca2+ concentrations. Similar to the positive KCa channel-gating modulators 1-ethyl-2- benzimidazolinone (1-EBIO) and cyclohexyl-[2-(3,5-dimethylpyrazol-1-yl)-6- methylpyrimidin-4-yl]-amine (CyPPA), the binding site for NS8593 has been assumed to be located in the C-terminal region, in which these channels interact with their Ca2+ sensor calmodulin. However, by using a progressive chimeric approach, we were able to localize the site-of-action of NS8593 to the KCa2 pore. For example, when we transferred the C terminus from the NS8593-insensitive intermediate-conductance KCa3.1 channel to K Ca2.3, the chimeric channel remained as sensitive to NS8593 as wild-type KCa2.3. In contrast, when we transferred the K Ca2.3 pore to KCa3.1, the channel became sensitive to NS8593. Using site-directed mutagenesis, we subsequently identified two specific residues in the inner vestibule of KCa2.3 (Ser507 and Ala532) that determined the effect of NS8593. Mutation of these residues to the corresponding residues in KCa3.1 (Thr250 and Val275) made KCa2.3 insensitive to NS8593, whereas introduction of serine and alanine into K Ca3.1 was sufficient to render this channel highly sensitive to NS8593. It is noteworthy that the same two residue positions have been found previously to mediate sensitivity of KCa3.1 to clotrimazole and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34). The location of Ser507 in the poreloop near the selectivity filter and Ala532 in an adjacent position in S6 are within the region predicted to contain the KCa2 channel gate. Hence, we propose that NS8593-mediated gating modulation occurs via interaction with gating structures at a position deep within the inner pore vestibule.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 899-909 |
| Number of pages | 11 |
| Journal | Molecular Pharmacology |
| Volume | 79 |
| Issue number | 6 |
| DOIs | |
| State | Published - Jun 2011 |
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ASJC Scopus subject areas
- Pharmacology
- Molecular Medicine
Cite this
Negative gating modulation by (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro- 1-naphthylamine (NS8593) depends on residues in the inner pore vestibule : Pharmacological evidence of deep-pore gating of KCa2 channels. / Jenkins, David Paul; Strøbæk, Dorte; Hougaard, Charlotte; Jensen, Marianne L.; Hummel, Rene; Sørensen, Ulrik S.; Christophersen, Palle; Wulff, Heike.
In: Molecular Pharmacology, Vol. 79, No. 6, 06.2011, p. 899-909.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Negative gating modulation by (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro- 1-naphthylamine (NS8593) depends on residues in the inner pore vestibule
T2 - Pharmacological evidence of deep-pore gating of KCa2 channels
AU - Jenkins, David Paul
AU - Strøbæk, Dorte
AU - Hougaard, Charlotte
AU - Jensen, Marianne L.
AU - Hummel, Rene
AU - Sørensen, Ulrik S.
AU - Christophersen, Palle
AU - Wulff, Heike
PY - 2011/6
Y1 - 2011/6
N2 - Acting as a negative gating modulator, (R)-N-(benzimidazol-2-yl)-1,2,3,4- tetrahydro-1-naphthylamine (NS8593) shifts the apparent Ca2+- dependence of the small-conductance Ca2+-activated K+ channels KCa2.1-2.3 to higher Ca2+ concentrations. Similar to the positive KCa channel-gating modulators 1-ethyl-2- benzimidazolinone (1-EBIO) and cyclohexyl-[2-(3,5-dimethylpyrazol-1-yl)-6- methylpyrimidin-4-yl]-amine (CyPPA), the binding site for NS8593 has been assumed to be located in the C-terminal region, in which these channels interact with their Ca2+ sensor calmodulin. However, by using a progressive chimeric approach, we were able to localize the site-of-action of NS8593 to the KCa2 pore. For example, when we transferred the C terminus from the NS8593-insensitive intermediate-conductance KCa3.1 channel to K Ca2.3, the chimeric channel remained as sensitive to NS8593 as wild-type KCa2.3. In contrast, when we transferred the K Ca2.3 pore to KCa3.1, the channel became sensitive to NS8593. Using site-directed mutagenesis, we subsequently identified two specific residues in the inner vestibule of KCa2.3 (Ser507 and Ala532) that determined the effect of NS8593. Mutation of these residues to the corresponding residues in KCa3.1 (Thr250 and Val275) made KCa2.3 insensitive to NS8593, whereas introduction of serine and alanine into K Ca3.1 was sufficient to render this channel highly sensitive to NS8593. It is noteworthy that the same two residue positions have been found previously to mediate sensitivity of KCa3.1 to clotrimazole and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34). The location of Ser507 in the poreloop near the selectivity filter and Ala532 in an adjacent position in S6 are within the region predicted to contain the KCa2 channel gate. Hence, we propose that NS8593-mediated gating modulation occurs via interaction with gating structures at a position deep within the inner pore vestibule.
AB - Acting as a negative gating modulator, (R)-N-(benzimidazol-2-yl)-1,2,3,4- tetrahydro-1-naphthylamine (NS8593) shifts the apparent Ca2+- dependence of the small-conductance Ca2+-activated K+ channels KCa2.1-2.3 to higher Ca2+ concentrations. Similar to the positive KCa channel-gating modulators 1-ethyl-2- benzimidazolinone (1-EBIO) and cyclohexyl-[2-(3,5-dimethylpyrazol-1-yl)-6- methylpyrimidin-4-yl]-amine (CyPPA), the binding site for NS8593 has been assumed to be located in the C-terminal region, in which these channels interact with their Ca2+ sensor calmodulin. However, by using a progressive chimeric approach, we were able to localize the site-of-action of NS8593 to the KCa2 pore. For example, when we transferred the C terminus from the NS8593-insensitive intermediate-conductance KCa3.1 channel to K Ca2.3, the chimeric channel remained as sensitive to NS8593 as wild-type KCa2.3. In contrast, when we transferred the K Ca2.3 pore to KCa3.1, the channel became sensitive to NS8593. Using site-directed mutagenesis, we subsequently identified two specific residues in the inner vestibule of KCa2.3 (Ser507 and Ala532) that determined the effect of NS8593. Mutation of these residues to the corresponding residues in KCa3.1 (Thr250 and Val275) made KCa2.3 insensitive to NS8593, whereas introduction of serine and alanine into K Ca3.1 was sufficient to render this channel highly sensitive to NS8593. It is noteworthy that the same two residue positions have been found previously to mediate sensitivity of KCa3.1 to clotrimazole and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34). The location of Ser507 in the poreloop near the selectivity filter and Ala532 in an adjacent position in S6 are within the region predicted to contain the KCa2 channel gate. Hence, we propose that NS8593-mediated gating modulation occurs via interaction with gating structures at a position deep within the inner pore vestibule.
UR - http://www.scopus.com/inward/record.url?scp=79956327044&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=79956327044&partnerID=8YFLogxK
U2 - 10.1124/mol.110.069807
DO - 10.1124/mol.110.069807
M3 - Article
C2 - 21363929
AN - SCOPUS:79956327044
VL - 79
SP - 899
EP - 909
JO - Molecular Pharmacology
JF - Molecular Pharmacology
SN - 0026-895X
IS - 6
ER -