Functional comparison of the K+-Cl- cotransporters KCC1 and KCC4

The K+-Cl- cotransporters (KCCs) are members of the cation-chloride cotrans porter gene family and fall into two phylogenetic subgroups: KCC2 paired wi th KCC4 and KCC1 paired with KCC3. We report a functional comparison in Xen opus oocytes of KCC1 and KCC4, widely expressed representatives of these tw o sub groups. KCC1 and KCC4 exhibit differential sensitivity to transport i nhibitors, such that KCC4 is much less sensitive to bumetanide and furosemi de. The efficacy of these anion inhibitors is critically dependent on the c oncentration of extracellular K+, with much higher inhibition in 50 mM K+ v ersus 2 mM K+. KCC4 is also uniquely sensitive to 10 mM barium and to 2 mM trichlormethiazide, Kinetic characterization reveals divergent affinities f or K+ (K-m values of similar to 25.5 and 17.5 mM for KCC1 and KCC4, respect ively), probably due to variation within the second transmembrane segment. Although the two isoforms have equivalent affinities for Cl-, they differ i n 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+-C1- 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 ab rogates hypotonic activation, an unexpected dissociation of N-ethylmaleimid e sensitivity and volume sensitivity. Although KCC4 is consistently more vo lume-sensitive, the hypotonic activation of both isoforms is critically dep endent on protein phosphatase 1. Overall, the functional comparison of thes e cloned K+-Cl- cotransporters reveals important functional, pharmacologica l, and kinetic differences with both physio logical and mechanistic implica tions.