Biochemical characterization of <i>hv</i>KCC and <i>hv</i>N(K)CC.

<p><i>Hv</i>KCC and <i>hv</i>N(K)CC were transiently (A, B) or stably transfected (C) in HEK293 cells. (A) Immunoblot analysis revealed a molecular mass of <i>hv</i>KCC and <i>hv</i>(N)KCC ranging from 115 to 215 kDa and 115 to 185 kDa, respectively. (B) Immunocytochemical analyses yielded subcellular distributions of <i>hv</i>KCC and <i>h</i>vNKCC similar to their mammalian counterparts (scale bar: 20 μm). (C) Cell surface expression analyses show that <i>hv</i>KCC (HEK^<i>hv</i>KCC, 15.1 ± 0.5%) and <i>hv</i>N(K)CC (HEK^{<i>hv</i>N(K)CC}, 10.3 ± 06%) are expressed at the cell membrane to a similar extent as <i>rn</i>KCC2 (HEK^{<i>rn</i>KCC2}, 11.8 ± 1.4%).</p

Molecular cloning and biochemical characterization of two cation chloride cotransporter subfamily members of <i>Hydra vulgaris</i>

<div><p>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 <i>Hydra vulgaris</i>. <i>Hv</i>KCC is composed of 1,032 amino-acid residues. Functional analyses revealed that <i>hv</i>KCC mediates a Na⁺-independent, Cl⁻ and K⁺ (Tl⁺)-dependent cotransport. The classification of <i>hv</i>KCC 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. <i>Hv</i>N(K)CC is composed of 965 amino-acid residues. Phylogenetic analyses and structural organization suggest that <i>hv</i>N(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, <i>hv</i>N(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.</p></div

Two-dimensional structural organization of <i>Hydra vulgaris</i> KCC and N(K)CC.

<p>Putative topology of <i>hv</i>KCC (A) and <i>hv</i>N(K)CC (B) determined by using the TOPCONS program (<a href="http://topcons.cbr.su.se/" target="_blank">http://topcons.cbr.su.se/</a>). The grey box represents the membrane containing the 12 predicted TMs. Branched lines between TMs 5 + 6 indicate N-glycosylation sites which were predicted using the program GlycoEP (<a href="http://www.imtech.res.in/raghava/glycoep/submit.html" target="_blank">http://www.imtech.res.in/raghava/glycoep/submit.html</a>).</p

Thallium transport activity of <i>hv</i>KCC and mammalian KCC2.

<p>HEK293 were stably transfected with <i>rn</i>KCC2 or <i>hv</i>KCC. Thallium flux measurements revealed that <i>hv</i>KCC exhibits a Cl⁻-dependent, Na⁺-independent inwardly directed transport of K⁺ (Tl⁺ as a congener) that is 1.4 times higher than background. *:p < 0.05; **:p < 0.005; ***p < 0.001; n = 6.</p

Primer list for cloning of <i>hv</i>KCC and <i>hv</i>N(K)CC.

<p>Primer list for cloning of <i>hv</i>KCC and <i>hv</i>N(K)CC.</p

Data_Sheet_1_K+/Cl− cotransporter 2 (KCC2) and Na+/HCO3− cotransporter 1 (NBCe1) interaction modulates profile of KCC2 phosphorylation.PDF

K+/Cl− cotransporter 2 (KCC2) is a major Cl− extruder in mature neurons and is responsible for the establishment of low intracellular [Cl−], necessary for fast hyperpolarizing GABAA-receptor mediated synaptic inhibition. Electrogenic sodium bicarbonate cotransporter 1 (NBCe1) is a pH regulatory protein expressed in neurons and glial cells. An interactome study identified NBCe1 as a possible interaction partner of KCC2. In this study, we investigated the putative effect of KCC2/NBCe1 interaction in baseline and the stimulus-induced phosphorylation pattern and function of KCC2. Primary mouse hippocampal neuronal cultures from wildtype (WT) and Nbce1-deficient mice, as well as HEK-293 cells stably transfected with KCC2WT, were used. The results show that KCC2 and NBCe1 are interaction partners in the mouse brain. In HEKKCC2 cells, pharmacological inhibition of NBCs with S0859 prevented staurosporine- and 4-aminopyridine (4AP)-induced KCC2 activation. In mature cultures of hippocampal neurons, however, S0859 completely inhibited postsynaptic GABAAR and, thus, could not be used as a tool to investigate the role of NBCs in GABA-dependent neuronal networks. In Nbce1-deficient immature hippocampal neurons, baseline phosphorylation of KCC2 at S940 was downregulated, compared to WT, and exposure to staurosporine failed to reduce pKCC2 S940 and T1007. In Nbce1-deficient mature neurons, baseline levels of pKCC2 S940 and T1007 were upregulated compared to WT, whereas after 4AP treatment, pKCC2 S940 was downregulated, and pKCC2 T1007 was further upregulated. Functional experiments showed that the levels of GABAAR reversal potential, baseline intracellular [Cl−], Cl− extrusion, and baseline intracellular pH were similar between WT and Nbce1-deficient neurons. Altogether, our data provide a primary description of the properties of KCC2/NBCe1 protein-protein interaction and implicate modulation of stimulus-mediated phosphorylation of KCC2 by NBCe1/KCC2 interaction—a mechanism with putative pathophysiological relevance.</p