Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs

Cation-chloride-cotransporters (CCCs) catalyze transport of Cl- with K+ and/or Na+ across cellular membranes. CCCs play roles in volume regulation, neural development and function, audition, blood pressure regulation, and renal function. CCCs are targets of drugs including loop diuretics and their disruption is implicated in pathophysiologies including epilepsy, hearing loss, and the genetic disorders Andermann, Gitelman, and Bartter syndromes. Here we present the cryo-EM structure of a CCC, the Mus musculus K+-Cl- cotransporter KCC4, in lipid nanodiscs. The structure, captured in an inside-open conformation, reveals the architecture of KCCs including an extracellular domain poised to regulate transport activity through an outer gate. We further identify substrate K+ and Cl- binding sites and provide a structural explanation for differences in substrate specificity and transport ratio between CCCs. These results provide mechanistic insight into the function and regulation of a physiologically important transporter family.