Abstract
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.
Highlights
The electroneutral cotransport of Kϩ and ClϪ across the plasma membrane is mediated by four members of the SLC12 cation-chloride cotransporter gene family, namely KCC1 [1], KCC2 [2], KCC3 [3,4,5], and KCC4 [3] (SLC12A4 –A7, respectively)
Isotonic Activity Is Conferred by the C-terminal KCC2 Unique Region—The ability of a given segment of KCC2 to confer isotonic transport in KCC4-KCC2 chimeras was studied using ClϪ-dependent 86Rbϩ uptake in Xenopus oocytes, measured under both isotonic and hypotonic conditions
The two major subtypes of cation-chloride cotransport, Naϩ-Kϩ2ClϪ and Kϩ-ClϪ cotransport, have opposing physiological roles that are subject to reciprocal regulation
Summary
Construction of Chimeric and Mutant cDNAs—For the purpose of constructing chimeric cDNAs between mouse KCC4 and human KCC2, we generated a number of silent restriction sites in the relevant cDNA constructs, all in the Xenopus expression vector pGEMHE [3, 9]. To generate a silent EcoRI site within transmembrane domain 12 (TM12) of mouse KCC4 (codons 664 – 666), we utilized the sense primer 5Ј-GAGTGGGGGGATGGAATTCGAGGCCTGTCACTGAAT-3Ј and antisense primer 5Ј-ATTCAGTGACAGGCCTCGAATTCCATCCCCCCACTC-3Ј. In the K4SD-2 chimera the region between the SfuI and MunI sites was replaced by KCC4, removing the first PEST domain and retaining the distal two-thirds of the unique region. In the K4SD-3 chimera, the region between the SfuI and BamHI sites was replaced by KCC4, retaining the distal one-third of the unique region of KCC2 (Fig. 2). Surface biotinylation was assessed, as a measure of protein expression at the cell membrane For this purpose, groups of 20 oocytes were injected with 25 ng of KCC cRNA and kept at 16 °C for 4 –5 days in ND96 medium. The pellet of streptavidin beads was resuspended in 65 l of sample buffer containing 4% -mercaptoethanol, heated at 50 °C for 20 min, and subjected to 7.5% SDS-PAGE and Western blot analysis with the KCC4specific antibody [43]
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