6,7-Dichloro-1H-indole-2,3-dione-3-oxime functions as a superagonist for the intermediate-conductance Ca2+-activated K+ channel KCa3.1.

Abstract

NS309 (6,7-dichloro-1H-indole-2,3-dione-3-oxime) is widely used as a pharmacological tool to increase the activity of small- and intermediate-conductance calcium-activated potassium channels. NS309 is assumed to function as a positive allosteric gating modulator. However, its binding site and the molecular details of its action remain unknown. Here, we show that NS309 has a profound effect on the calcium-dependent gating of the intermediate-conductance Ca²⁺-activated K⁺ channel K_Ca3.1. In inside-out experiments, 10 μM NS309 shifted the calcium EC₅₀ from 430 to 31 nM. In whole-cell experiments, changing free intracellular calcium from 250 nM to 3 μM decreased the EC₅₀ of NS309 from 74 to 8.6 nM. We further observed that NS309 could elicit greater responses than saturating calcium, making it a "superagonist." Molecular modeling suggested 2 possible binding sites for NS309 in K_Ca3.1, which we probed by mutagenesis and determined that NS309 is binding in the interface between the S₄₅A segment of the intracellular S4-S5 linker and the N-lobe of the channel-associated calmodulin. Molecular dynamic simulations revealed that NS309 pushes several water molecules out of the interface pocket, establishes stable contacts with S181 and L185 in the S₄₅A segment of K_Ca3.1 and E54 in calmodulin, and promotes longer sustained widening of the inner gate of K_Ca3.1 at V282 in the S6 segment. Polar substitutions of the hydrophobic-gating residues V282 and A279 resulted in constitutively open channels that could not be further potentiated by NS309, suggesting that NS309 produces its agonistic effects by increasing the open probability of the inner gate of K_Ca3.1. SIGNIFICANCE STATEMENT: The publication of the full-length cryo-electron microscopy structure of the intermediate-conductance Ca²⁺-activated K⁺ channel K_Ca3.1 suggested that the previously reported binding site of NS309 (6,7-dichloro-1H-indole-2,3-dione-3-oxime) was a crystallization artifact because this structure only included the C-terminus and the channel-associated calmodulin. This study demonstrates that the true binding site of NS309 is located between the S4 and S5 linker of K_Ca3.1 and the N-lobe of calmodulin. NS309 acts as a stabilizing force within the gating interface and increases the open probability of the inner hydrophobic gate.