The K⁺-Cl⁻cotransporters (KCCs) belong to the gene family of electroneutral cation-chloride cotransporters, which also includes two bumetanide-sensitive Na⁺-K⁺-2Cl⁻cotransporters and a thiazide-sensitive Na⁺-Cl⁻ cotransporter. We have cloned cDNAs encoding mouse KCC3, human KCC3, and human KCC4, three new members of this gene family. The KCC3 and KCC4 cDNAs predict proteins of 1083 and 1150 amino acids, respectively. The KCC3 and KCC4 proteins are 65–71% identical to the previously characterized transporters KCC1 and KCC2, with which they share a predicted membrane topology. The four KCC proteins differ at amino acid residues within key transmembrane domains and in the distribution of putative phosphorylation sites within the amino- and carboxyl-terminal cytoplasmic domains. The expression of mouse KCC3 in Xenopus laevis oocytes reveals the expected functional characteristics of a K⁺Cl⁻ cotransporter: Cl⁻-dependent uptake of⁸⁶Rb⁺ which is strongly activated by cell swelling and weakly sensitive to furosemide. A direct functional comparison of mouse KCC3 to rabbit KCC1 indicates that KCC3 has a much greater volume sensitivity. The human KCC3 and KCC4 genes are located on chromosomes 5p15 and 15q14, respectively. Although widely expressed, KCC3 transcripts are the most abundant in heart and kidney, and KCC4 is expressed in muscle, brain, lung, heart, and kidney. The unexpected molecular heterogeneity of K⁺-Cl⁻ cotransport has implications for the physiology and pathophysiology of a number of tissues.

We report the molecular and functional characterization of murine Slc26a6, the putative apical chloride-formate exchanger of the proximal tubule. The Slc26a6 transcript is expressed in several tissues, including kidney. Alternative splicing of the second exon generates two distinct isoforms, denoted Slc26a6a and Slc26a6b, which differ in the inclusion of a 23-residue NH₂-terminal extension. Functional comparison with murine Slc26a1, the basolateral oxalate exchanger of the proximal tubule, reveals a number of intriguing differences. Whereas Slc26a6 is capable of Cl⁻, SO[ ], formate, and oxalate uptake when expressed in Xenopus laevis oocytes, Slc26a1 transports only SO[ ] and oxalate. Measurement of intracellular pH during the removal of extracellular Cl⁻in the presence and absence of HCO[ ] indicates that Slc26a6 functions as both a Cl⁻/HCO[ ] and a Cl⁻/OH⁻exchanger; simultaneous membrane hyperpolarization during these experimental maneuvers reveals that HCO[ ] and OH⁻transport mediated by Slc26a6 is electrogenic. Cis-inhibition and efflux experiments indicate that Slc26a6 can mediate the exchange of both Cl⁻and SO[ ]with a number of substrates, including formate and oxalate. In contrast, SO[ ] and oxalate transport by Slc26a1 are mutually cis-inhibited but activated significantly by extracellular halides, lactate, and formate. The data indicate that Slc26a6 encodes an apical Cl⁻/formate/oxalate and Cl⁻/base exchanger and reveal significant mechanistic differences between apical and basolateral oxalate exchangers of the proximal tubule.

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 inXenopus 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 agentN-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.

The neuron-specific K(+)-Cl(-) cotransporter KCC2 plays a crucial role in determining intracellular chloride activity and thus the neuronal response to gamma-aminobutyric acid and glycine. Of the four KCCs, KCC2 is unique in mediating constitutive K(+)-Cl(-) cotransport under isotonic conditions; the other three KCCs are exclusively swelling-activated, with no isotonic activity. We have utilized a series of chimeric cDNAs to localize the determinant of isotonic transport in KCC2. Two generations of chimeric KCC4-KCC2 cDNAs initially localized this characteristic to within a KCC2-specific expansion of the cytoplasmic C terminus, between residues 929 and 1043. This region of KCC2 is rich in prolines, serines, and charged residues and encompasses two predicted PEST sequences. Substitution of this region in KCC2 with the equivalent sequence of KCC4 resulted in a chimeric KCC that was devoid of isotonic activity, with intact swelling-activated transport. A third generation of chimeras demonstrated that a domain just distal to the PEST sequences confers isotonic transport on KCC4. Mutagenesis of this region revealed that residues 1021-1035 of KCC2 are sufficient for isotonic transport. Swelling-activated K(+)-Cl(-) cotransport is abrogated by calyculin A, whereas isotonic transport mediated by KCC chimeras and KCC2 is completely resistant to this serine-threonine phosphatase inhibitor. In summary, a 15-residue C-terminal domain in KCC2 is both necessary and sufficient for constitutive K(+)-Cl(-) cotransport under isotonic conditions. Furthermore, unlike swelling-activated transport, constitutive K(+)-Cl(-) cotransport mediated by KCC2 is completely independent of serine-threonine phosphatase activity, suggesting that these two modes of transport are activated by distinct mechanisms.