An Amino Acid Outside the Pore Region Influences Apamin Sensitivity in Small Conductance Ca2+-activated K+ Channels

Abstract

Small conductance calcium-activated potassium channels (SK, K Ca ) are a family of voltageindependent K + channels with a distinct physiology and pharmacology. The bee venom toxin apamin inhibits exclusively the three cloned SK channel subtypes (SK1, SK2 and SK3) with different affinity, highest for SK2, lowest for SK1 and intermediate for SK3 channels. The high selectivity of apamin made it a valuable tool to study the molecular makeup and function of native SK channels. Three amino acids located in the outer vestibule of the pore are of particular importance for the different apamin sensitivities of SK channels. Chimeric SK1 channels, enabling the homomeric expression of the rat SK1 (rSK1) subunit and containing the core domain (S1-S6) of rSK1, are apamin insensitive. By contrast, channels formed by the human orthologue hSK1 are sensitive to apamin. This finding hinted to the involvement of regions beyond the pore as determinants of apamin sensitivity, since hSK1 and rSK1 have an identical amino acid sequence in the pore region. Here we investigated which parts of the channels outside the pore region are important for apamin sensitivity by constructing chimeras between apamin insensitive and sensitive SK channel subunits and by introducing point mutations. We demonstrate that a single amino acid situated in the extracellular loop between the transmembrane segments S3 and S4 has a major impact on apamin sensitivity. Our findings enabled us to convert the hSK1 channel into a channel that was as sensitive for apamin as SK2, the SK channel with the highest sensitivity. Ca 2+ -activated K + channels (K Ca ) 1 are activated by rises in intracellular Ca 2+ . The K Ca potassium channel family comprises of at least three subfamilies, K Ca 1-3 (1). Channels containing the K Ca 1.1 α-subunit (BK channels) have large single channel conductance and are maximally activated by micromolar concentrations of intracellular free calcium and concurrent depolarization (2). Their kinetic and pharmacological properties are modified upon assembly with membrane standing ß-subunits (3). The K Ca 2 subfamily of small-conductance Ca 2+ -activated K + channels, also known as SK channels, has three closely related members SK1 (K Ca 2.1), SK2 (K Ca 2.2) and SK3 (K Ca 2.3), which are characterized by a small single channel conductance. The IK channel (K Ca 3.1) shows an intermediate single channel conductance. Both SK and IK channels are voltage independent and activated by submicromolar concentrations of intracellular free Ca 2+ . The gating of SK and IK channels is induced upon Ca 2+ binding to calmodulin, which is constitutively bound to each channel subunit. Ca 2+ * This work was supported by a Wellcome Prize studentship (T.F.) and a Wellcome Trust Senior Research fellowship (M.S.).Address correspondence to: Martin Stocker, Laboratory of Molecular Pharmacology, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK, UKPMC Funders Group Author Manuscript UKPMC Funders Group Author Manuscriptbindin...

Crucial role of a shared extracellular loop in apamin sensitivity and maintenance of pore shape of small-conductance calcium-activated potassium (SK) channels

“…Block of SK channel current by apamin is clearly more complicated than these data suggest, as despite containing an identical outer pore sequence to the apamin-sensitive hSK1 and a serine in this position in the S3-S4 loop, rSK1 is apamin insensitive. Replacement of the S3-S4 loop of rSK1 with that of hSK1 produced a channel with a sensitivity to apamin that was similar to that of hSK1 (8). These data indicate that an additional interaction site(s) within the S3-S4 extracellular loop contributes to binding or block by apamin.…”

“…The toxin is most potent at SK2 (IC 50 ∼ 70 pM) (5-9) and SK3 (IC 50 ∼ 0.63-6 nM) (7, 9, 10), followed by the human isoform of SK1 (IC 50 ∼ 1-8 nM) (8, 9), whereas the rat isoform of SK1 is apamin insensitive (11,12). Mutation studies have identified a number of residues within the outer pore that affect block by apamin (7,8,13). For example, an outer pore histidine residue has been shown to be critical for both binding and block by the toxin (7).…”

“…These data suggested that apamin does not behave as a classical pore blocker, but blocks using an allosteric mechanism (7). Apamin also interacts with a serine residue in the S3-S4 extracellular loop to impart a highsensitivity block of SK2, with mutation of the corresponding threonine in hSK1 to a serine increasing sensitivity of block of hSK1 by the toxin (8). Block of SK channel current by apamin is clearly more complicated than these data suggest, as despite containing an identical outer pore sequence to the apamin-sensitive hSK1 and a serine in this position in the S3-S4 loop, rSK1 is apamin insensitive.…”

Physiologie, pharmacologie et modélisation de canaux potassiques

“…La pré-sence d'un résidu histidine conservé dans les trois sous-types et localisé dans la tourelle en position 490 (numérotation hSK3) s'est avérée essentielle pour le blocage des canaux par la toxine [21,49]. D'autres études complémentaires ont récemment montré que des résidus éloignés de la région du pore étaient également impliqués dans le blocage du courant par l'apamine [31,50], renforçant l'idée d'une action allostérique. Cette conclusion est confortée par le fait que les IC 50 de SK2 et SK3 sont différentes mais que leurs K d sont identiques.…”

“…Cet agent pré-sente une affinité pour les canaux SK (Ki ≈ 0,3 μM) environ cinq fois supérieure à celle de la NML (Ki ≈ 1,6 μM) et pratiquement équiva-lente à celle du déqualinium (Ki ≈ 0,2 μM). D'autre part, l'AG525E1 bloque de manière plus ou moins équivalente les trois sous-types de canaux SK (IC 50 • L'autre objectif important est de développer des bloqueurs présen-tant une sélectivité vis-à-vis des canaux SK assez grande pour être exploitable (elle doit être au moins 100 fois plus puissante pour l'un des sous-types). Jusqu'à présent, cette sélectivité n'a été démontrée que pour des ligands peptidiques.…”