Functional Comparison of the K⁺-Cl⁻Cotransporters KCC1 and KCC4*

The K⁺-Cl⁻cotransporters (KCCs) are members of the cation-chloride cotransporter gene family and fall into two phylogenetic subgroups: KCC2 paired with KCC4 and KCC1 paired with KCC3. We report a functional comparison in Xenopus oocytes of KCC1 and KCC4, widely expressed representatives of these two subgroups. KCC1 and KCC4 exhibit differential sensitivity to transport inhibitors, such that KCC4 is much less sensitive to bumetanide and furosemide. The efficacy of these anion inhibitors is critically dependent on the concentration of extracellular K⁺, with much higher inhibition in 50 mm K⁺ versus 2 mmK⁺. KCC4 is also uniquely sensitive to 10 mmbarium and to 2 mm trichlormethiazide. Kinetic characterization reveals divergent affinities for K⁺(K_m values of ∼25.5 and 17.5 mm for KCC1 and KCC4, respectively), probably due to variation within the second transmembrane segment. Although the two isoforms have equivalent affinities for Cl⁻, they differ in the anion selectivity of K⁺ transport (Cl⁻ > SCN⁻ = Br⁻ > PO₄⁻³ > I⁻for KCC1 and Cl⁻ > Br⁻ > PO₄⁻³ = I⁻ > SCN⁻for KCC4). Both KCCs express minimal K⁺-Cl⁻cotransport under isotonic conditions, with significant activation by cell swelling under hypotonic conditions. The cysteine-alkylating agent N-ethylmaleimide activates K⁺-Cl⁻ cotransport in isotonic conditions but abrogates hypotonic activation, an unexpected dissociation of N-ethylmaleimide sensitivity and volume sensitivity. Although KCC4 is consistently more volume-sensitive, the hypotonic activation of both isoforms is critically dependent on protein phosphatase 1. Overall, the functional comparison of these cloned K⁺-Cl⁻ cotransporters reveals important functional, pharmacological, and kinetic differences with both physiological and mechanistic implications.