bioRxiv2026
Hyponatremia is a prevalent disorder marked by excess water retention and substantial morbidity, motivating interest in the CLC-Ka chloride channel as a therapeutic target. Selectively inhibiting CLC-Ka without affecting the closely related CLC-Kb is essential for preventing serious side effects. However, developing isoform-selective inhibitors has been challenging because most small molecules do not distinguish between CLC-Ka and CLC-Kb, and the basis for selectivity in the few known exceptions remains unclear. The small molecule BIM1 preferentially inhibits CLC-Ka over CLC-Kb, providing an opportunity to dissect isoform-specific pharmacology. To investigate this mechanism, we determined cryo-EM structures of BIM1 and BIM15, a related nonselective analog, bound to a CLC-K variant engineered to match the human CLC-Ka binding pocket. Structural and computational analyses reveal that inhibition and isoform selectivity are anchored by interactions with a conserved lysine, with surrounding binding-site residues subtly tuning the local electrostatic environment to promote or disfavor these contacts. These analyses further identify a dynamic extracellular loop that intermittently occludes the shared pathway accessing the inhibitor-binding site and pore. Bound BIM15 engages this gating loop more extensively than BIM1, suggesting that differential loop engagement contributes to inhibitor selectivity, a prediction validated by mutagenesis. Because loop dynamics block the pore, we examined the structural impact of Ca{superscript 2}, which favors opening, and found the gating loop ordered and withdrawn from the pathway. Together, these findings define how binding-site microenvironments and gating-loop dynamics shape isoform-specific inhibition and pore access in CLC-K channels. Significance StatementHyponatremia is a major clinical problem with limited therapeutic options. The kidney chloride channel CLC-Ka is an attractive drug target, but its high sequence identity to CLC-Kb has hindered the development of isoform-selective inhibitors needed for safe therapy. A low-micromolar CLC-Ka-selective inhibitor had been identified, providing a foothold for drug development, but the structural basis of its selectivity was unknown. Here, by integrating cryo-EM structures with molecular dynamics simulations, we define the inhibitor-binding site and reveal the mechanism that enables preferential CLC-Ka inhibition. We further show that a dynamic extracellular loop functions as a gating element shaping inhibitor access and engagement. These findings establish a mechanistic foundation for developing improved treatments for hyponatremia.