Apamin-sensitive small conductance calcium-activated potassium channels (SKCa1-3) mediate the slow afterhyperpolarization in neurons, but the molecular identity of the channel has not been defined because of the lack of specific inhibitors. Here we describe the structure-based design of a selective inhibitor of SKCa2. Leiurotoxin I (Lei) and PO5, peptide toxins that share the RXCQ motif, potently blocked human SKCa2 and SKCa3 but not SKCa1, whereas maurotoxin, Pi1, Tsκ, and PO1 were ineffective. Lei blocked these channels more potently than PO5 because of the presence of Ala1, Phe2, and Met7. By replacing Met7 in the RXCQ motif of Lei with the shorter, unnatural, positively charged diaminobutanoic acid (Dab), we generated Lei-Dab7, a selective SKCa2 inhibitor (Kd = 3.8 nM) that interacts with residues in the external vestibule of the channel. SKCa3 was rendered sensitive to Lei-Dab7 by replacing His521 with the corresponding SKCa2 residue (Asn 367). Intra-cerebroventricular injection of Lei-Dab7 into mice resulted in no gross central nervous system toxicity at concentrations that specifically blocked SKCa2 homotetramers. Lei-Dab7 will be a useful tool to investigate the functional role of SKCa2 in mammalian tissues.
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