Differences in the Large Extracellular Loop between the K+-Cl− Cotransporters KCC2 and KCC4

Cation Chloride Cotransporters (CCCs) comprise secondary active membrane proteins mainly mediating the symport of cations (Na+, K+) coupled with chloride (Cl−). They are divided into K+-Cl− outward transporters (KCCs), the Na+-K+-Cl− (NKCCs) and Na+-Cl− (NCCs) inward transporters, the cation chloride cotransporter interacting protein CIP1, and the polyamine transporter CCC9. KCCs and N(K)CCs are established in the genome since eukaryotes and metazoans, respectively. Most of the physiological and functional data were obtained from vertebrate species. To get insights into the basal functional properties of KCCs and N(K)CCs in the metazoan lineage, we cloned and characterized KCC and N(K)CC from the cnidarian Hydra vulgaris. HvKCC is composed of 1,032 amino-acid residues. Functional analyses revealed that hvKCC mediates a Na+-independent, Cl− and K+ (Tl+)-dependent cotransport. The classification of hvKCC as a functional K-Cl cotransporter is furthermore supported by phylogenetic analyses and a similar structural organization. Interestingly, recently obtained physiological analyses indicate a role of cnidarian KCCs in hyposmotic volume regulation of nematocytes. HvN(K)CC is composed of 965 amino-acid residues. Phylogenetic analyses and structural organization suggest that hvN(K)CC is a member of the N(K)CC subfamily. However, no inorganic ion cotransport function could be detected using different buffer conditions. Thus, hvN(K)CC is a N(K)CC subfamily member without a detectable inorganic ion cotransporter function. Taken together, the data identify two non-bilaterian solute carrier 12 (SLC12) gene family members, thereby paving the way for a better understanding of the evolutionary paths of this important cotransporter family.

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“…Uptake-measurements were done as previously described [47,48]. Either stable Flp-In cell lines or transiently transfected cells were used.…”

Section: Methodsmentioning

confidence: 99%

Molecular cloning and biochemical characterization of two cation chloride cotransporter subfamily members of Hydra vulgaris

et al. 2017

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“…First, the scaffold is followed by a C-terminal domain (CTD) important for regulating expression, trafficking, and activity including through phosphorylation or dephosphorylation of CTD sites in response to cell swelling 16–19 . Second, CCCs contain a “long extracellular loop” with predicted disulfide bonds and glycosylation sites that differs in position and structure between CCCs; it is formed by the region between TM5-TM6 in KCCs and between TM7-TM8 in NKCCs 14, 20 .…”

Section: Introductionmentioning

confidence: 99%

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

Reid

Kern

Brohawn

2019

Preprint

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AbstractCation-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.

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“…Construction of Expression Clones-Wild-type rat KCC2b (GenBank TM accession number NM_134363) and HA-tagged mouse KCC2b (GenBank TM accession number NM_020333) expression clones with an HA tag at the N terminus or in the second extracellular loop were reported previously (47,48). Site-directed mutagenesis of KCC2b cDNA was performed according to the QuikChange mutagenesis system (Stratagene, Heidelberg, Germany).…”

Section: ϫ2mentioning

confidence: 99%

“…Uptake measurements were done as described previously (49,50,47). Cells were transiently transfected with the respective construct, using TurboFect (Fermentas, Schwerte, Germany), according to the protocol provided.…”

Section: ϫ2mentioning

confidence: 99%

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A Novel Regulatory Locus of Phosphorylation in the C Terminus of the Potassium Chloride Cotransporter KCC2 That Interferes with N-Ethylmaleimide or Staurosporine-mediated Activation*♦

Weber

Hartmann

Beyer

et al. 2014

Journal of Biological Chemistry

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Differences in the Large Extracellular Loop between the K+-Cl− Cotransporters KCC2 and KCC4

Cited by 36 publications

References 41 publications

Molecular cloning and biochemical characterization of two cation chloride cotransporter subfamily members of Hydra vulgaris

Molecular cloning and biochemical characterization of two cation chloride cotransporter subfamily members of Hydra vulgaris

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

A Novel Regulatory Locus of Phosphorylation in the C Terminus of the Potassium Chloride Cotransporter KCC2 That Interferes with N-Ethylmaleimide or Staurosporine-mediated Activation*♦

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