Dataset Information

Structural Determinants for the Selectivity of the Positive KCa3.1 Gating Modulator 5-Methylnaphtho[2,1-d]oxazol-2-amine (SKA-121).

ABSTRACT: Intermediate-conductance (KCa3.1) and small-conductance (KCa2) calcium-activated K+ channels are gated by calcium binding to calmodulin (CaM) molecules associated with the calmodulin-binding domain (CaM-BD) of these channels. The existing KCa activators, such as naphtho[1,2-d]thiazol-2-ylamine (SKA-31), 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309), and 1-ethylbenzimidazolin-2-one (EBIO), activate both channel types with similar potencies. In a previous chemistry effort, we optimized the benzothiazole pharmacophore of SKA-31 toward KCa3.1 selectivity and identified 5-methylnaphtho[2,1-d]oxazol-2-amine (SKA-121), which exhibits 40-fold selectivity for KCa3.1 over KCa2.3. To understand why introduction of a single CH3 group in five-position of the benzothiazole/oxazole system could achieve such a gain in selectivity for KCa3.1 over KCa2.3, we first localized the binding site of the benzothiazoles/oxazoles to the CaM-BD/CaM interface and then used computational modeling software to generate models of the KCa3.1 and KCa2.3 CaM-BD/CaM complexes with SKA-121. Based on a combination of mutagenesis and structural modeling, we suggest that all benzothiazole/oxazole-type KCa activators bind relatively "deep" in the CaM-BD/CaM interface and hydrogen bond with E54 on CaM. In KCa3.1, SKA-121 forms an additional hydrogen bond network with R362. In contrast, NS309 sits more "forward" and directly hydrogen bonds with R362 in KCa3.1. Mutating R362 to serine, the corresponding residue in KCa2.3 reduces the potency of SKA-121 by 7-fold, suggesting that R362 is responsible for the generally greater potency of KCa activators on KCa3.1. The increase in SKA-121's KCa3.1 selectivity compared with its parent, SKA-31, seems to be due to better overall shape complementarity and hydrophobic interactions with S372 and M368 on KCa3.1 and M72 on CaM at the KCa3.1-CaM-BD/CaM interface.

SUBMITTER: Brown BM

PROVIDER: S-EPMC5588545 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Structural Determinants for the Selectivity of the Positive KCa3.1 Gating Modulator 5-Methylnaphtho[2,1-d]oxazol-2-amine (SKA-121).

Brown Brandon M BM Shim Heesung H Zhang Miao M Yarov-Yarovoy Vladimir V Wulff Heike H

Molecular pharmacology 20170731 4

Intermediate-conductance (KCa3.1) and small-conductance (KCa2) calcium-activated K+ channels are gated by calcium binding to calmodulin (CaM) molecules associated with the calmodulin-binding domain (CaM-BD) of these channels. The existing KCa activators, such as naphtho[1,2-d]thiazol-2-ylamine (SKA-31), 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309), and 1-ethylbenzimidazolin-2-one (EBIO), activate both channel types with similar potenc ...[more]

PMID: 28760780

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Project description:In this work we address the question of the KCa3.1 channel pore structure in the closed configuration in relation to the contribution of the C-terminal end of the S6 segments to the Ca(2+)-dependent gating process. Our results based on SCAM (substituted cysteine accessibility method) experiments first demonstrate that the S6 transmembrane segment of the open KCa3.1 channel contains two distinct functional domains delimited by V282 with MTSEA and MTSET binding leading to a total channel inhibition at positions V275, T278, and V282 and to a steep channel activation at positions A283 and A286. The rates of modification by MTSEA (diameter 4.6 A) of the 275C (central cavity) and 286C residues (S6 C-terminal end) for the closed channel configuration were found to differ by less than sevenfold, whereas experiments performed with the larger MTSET reagent (diameter 5.8 A) resulted in modification rates 10(3)-10(4) faster for cysteines at 286 compared with 275. Consistent with these results, the modification rates of the cavity lining 275C residue by MTSEA, Et-Hg(+), and Ag(+) appeared poorly state dependent, whereas modification rates by MTSET were 10(3) faster for the open than the closed configuration. A SCAM analysis of the channel inner vestibule in the closed state revealed in addition that cysteine residues at 286 were accessible to MTS reagents as large as MTS-PtrEA, a result supported by the observation that binding of MTSET to cysteines at positions 283 or 286 could neither sterically nor electrostatically block the access of MTSEA to the closed channel cavity (275C). It follows that the closed KCa3.1 structure can hardly be accountable by an inverted teepee-like structure as described for KcsA, but is better represented by a narrow passage centered at V282 (equivalent to V474 in Shaker) connecting the channel central cavity to the cytosolic medium. This passage would not be however restrictive to the diffusion of small reagents such as MTSEA, Et-Hg(+), and Ag(+), arguing against the C-terminal end of S6 forming an obstructive barrier to the diffusion of K(+) ions for the closed channel configuration.

Project description:Background and purposeThe calcium-activated potassium channel KCa3.1 is expressed in the vascular endothelium where its activation causes endothelial hyperpolarization and initiates endothelium-derived hyperpolarization (EDH)-dependent dilatation. Here, we investigated whether pharmacological activation of KCa3.1 dilates skeletal muscle arterioles and whether myoendothelial gap junctions formed by connexin40 (Cx40) are required for EDH-type dilatations and pressure depressor responses in vivo.Experimental approachWe performed intravital microscopy in the cremaster muscle microcirculation and blood pressure telemetry in Cx40-deficient mice.Key resultsIn wild-type mice, the KCa3.1-activator SKA-31 induced pronounced concentration-dependent arteriolar EDH-type dilatations, amounting to ?40% of maximal dilatation, and enhanced the effects of ACh. These responses were absent in mice devoid of KCa3.1 channels. In contrast, SKA-31-induced dilatations were not attenuated in mice with endothelial cells deficient in Cx40 (Cx40(fl/fl):Tie2-Cre). In isolated endothelial cell clusters, SKA-31 induced hyperpolarizations of similar magnitudes (by ?38 mV) in Cx40(fl/fl):Tie2-Cre, ubiquitous Cx40-deficient mice (Cx40(-/-)) and controls (Cx40(fl/fl)), which were reversed by the specific KCa3.1-blocker TRAM-34. In normotensive wild-type and Cx40(fl/fl):Tie2-Cre as well as in hypertensive Cx40(-/-) animals, i.p. injections of SKA-31 (30 and 100 mg·kg(-1)) decreased arterial pressure by ?32 mmHg in all genotypes. The depressor response to 100 mg·kg(-1) SKA-31 was associated with a decrease in heart rate.Conclusions and implicationsWe conclude that endothelial hyperpolarization evoked by pharmacological activation of KCa3.1 channels induces EDH-type arteriolar dilatations that are independent of endothelial Cx40 and Cx40-containing myoendothelial gap junctions. As SKA-31 reduced blood pressure in hypertensive Cx40-deficient mice, KCa3.1 activators may be useful drugs for severe treatment-resistant hypertension.

Dataset Information

Structural Determinants for the Selectivity of the Positive KCa3.1 Gating Modulator 5-Methylnaphtho[2,1-d]oxazol-2-amine (SKA-121).

Publications

Structural Determinants for the Selectivity of the Positive KCa3.1 Gating Modulator 5-Methylnaphtho[2,1-<i>d</i>]oxazol-2-amine (SKA-121).

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