Both chloride-binding sites are required for KCC2-mediated transport

The K+–Cl− cotransporter 2 (KCC2) plays an important role in inhibitory neurotransmission, and its impairment is associated with neurological and psychiatric disorders, including epilepsy, schizophrenia, and autism. Although KCCs transport K+ and Cl− in a 1:1 stoichiometry, two Cl− coordination sites were indicated via cryo-EM. In a comprehensive analysis, we analyzed the consequences of point mutations of residues coordinating Cl− in Cl1 and Cl2. Individual mutations of residues in Cl1 and Cl2 reduce or abolish KCC2WT function, indicating a crucial role of both Cl− coordination sites for KCC2 function. Structural changes in the extracellular loop 2 by inserting a 3xHA tag switches the K+ coordination site to another position. To investigate, whether the extension of the extracellular loop 2 with the 3xHA tag also affects the coordination of the two Cl− coordination sites, we carried out the analogous experiments for both Cl− coordinating sites in the KCC2HA construct. These analyses showed that most of the individual mutation of residues in Cl1 and Cl2 in the KCC2HA construct reduces or abolishes KCC2 function, indicating that the coordination of Cl− remains at the same position. However, the coupling of K+ and Cl− in Cl1 is still apparent in the KCC2HA construct, indicating a mutual dependence of both ions. In addition, the coordination residue Tyr569 in Cl2 shifted in KCC2HA. Thus, conformational changes in the extracellular domain affect K+ and Cl−-binding sites. However, the effect on the Cl−-binding sites is subtler. The K+–Cl− cotransporter 2 (KCC2) plays an important role in inhibitory neurotransmission, and its impairment is associated with neurological and psychiatric disorders, including epilepsy, schizophrenia, and autism. Although KCCs transport K+ and Cl− in a 1:1 stoichiometry, two Cl− coordination sites were indicated via cryo-EM. In a comprehensive analysis, we analyzed the consequences of point mutations of residues coordinating Cl− in Cl1 and Cl2. Individual mutations of residues in Cl1 and Cl2 reduce or abolish KCC2WT function, indicating a crucial role of both Cl− coordination sites for KCC2 function. Structural changes in the extracellular loop 2 by inserting a 3xHA tag switches the K+ coordination site to another position. To investigate, whether the extension of the extracellular loop 2 with the 3xHA tag also affects the coordination of the two Cl− coordination sites, we carried out the analogous experiments for both Cl− coordinating sites in the KCC2HA construct. These analyses showed that most of the individual mutation of residues in Cl1 and Cl2 in the KCC2HA construct reduces or abolishes KCC2 function, indicating that the coordination of Cl− remains at the same position. However, the coupling of K+ and Cl− in Cl1 is still apparent in the KCC2HA construct, indicating a mutual dependence of both ions. In addition, the coordination residue Tyr569 in Cl2 shifted in KCC2HA. Thus, conformational changes in the extracellular domain affect K+ and Cl−-binding sites. However, the effect on the Cl−-binding sites is subtler. Structural changes in the extracellular loop 2 of the murine KCC2 potassium chloride cotransporter modulate ion transportJournal of Biological ChemistryVol. 296PreviewK+–Cl− cotransporters (KCCs) play important roles in physiological processes such as inhibitory neurotransmission and cell-volume regulation. KCCs exhibit significant variations in K+ affinities, yet recent atomic structures demonstrated that K+- and Cl−-binding sites are highly conserved, raising the question of whether additional structural elements may contribute to ion coordination. The termini and the large extracellular domain (ECD) of KCCs exhibit only low sequence identity and were already discussed as modulators of transport activity. Full-Text PDF Open Access The amino acid/polyamine/organocation superfamily comprises the second largest superfamily of secondary carriers (1Wong F.H. Chen J.S. Reddy V. Day J.L. Shlykov M.A. Wakabayashi S.T. et al.The amino acid-polyamine-organocation superfamily.Microb. Physiol. 2012; 22: 105-113Crossref Scopus (84) Google Scholar), initially described by Saier et al. (2Jack D.L. Paulsen I.T. Saier M.H. The amino acid/polyamine/organocation (APC) superfamily of transporters specific for amino acids, polyamines and organocations.Microbiology. 2000; 146: 1797-1814Crossref PubMed Scopus (226) Google Scholar). Central to the superfamily members is the LeuT-fold (3Hediger M.A. Clémençon B. Burrier R.E. Bruford E.A. The ABCs of membrane transporters in health and disease (SLC series): introduction.Mol. Aspects Med. 2013; 34: 95-107Crossref PubMed Scopus (415) Google Scholar, 4Höglund P.J. Nordström K.J.V. Schiöth H.B. Frederiksson R. The solute carrier families have a remarkably long evolutionary history with the majority of the human families present before divergence of Bilaterian species.Mol. Biol. Evol. 2010; 28: 1531-1541Crossref PubMed Scopus (129) Google Scholar, 5Arroyo J.P. Kahle K.T. Gamba G. The SLC12 family of electroneutral cation-coupled chloride cotransporters.Mol. Aspects Med. 2013; 34: 288-298Crossref PubMed Scopus (105) Google Scholar), which is mainly characterized by a conserved five-helix transmembrane inverted repeat motif (5 + 5 topology). This fold is highly conserved within the amino acid/polyamine/organocation superfamily. A family within this superfamily is the cation chloride cotransporter (CCC) family (3Hediger M.A. Clémençon B. Burrier R.E. Bruford E.A. The ABCs of membrane transporters in health and disease (SLC series): introduction.Mol. Aspects Med. 2013; 34: 95-107Crossref PubMed Scopus (415) Google Scholar, 4Höglund P.J. Nordström K.J.V. Schiöth H.B. Frederiksson R. The solute carrier families have a remarkably long evolutionary history with the majority of the human families present before divergence of Bilaterian species.Mol. Biol. Evol. 2010; 28: 1531-1541Crossref PubMed Scopus (129) Google Scholar, 5Arroyo J.P. Kahle K.T. Gamba G. The SLC12 family of electroneutral cation-coupled chloride cotransporters.Mol. Aspects Med. 2013; 34: 288-298Crossref PubMed Scopus (105) Google Scholar). The CCCs consist of secondary active membrane transporters that mediate the symport of cations (Na+ and K+) coupled with chloride (Cl−) (6Gamba G. Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporter.Physiol. Rev. 2005; 85: 423-493Crossref PubMed Scopus (654) Google Scholar). Gene duplication events at the base of archaeans and eukaryotes led to the diversification and neofunctionalization of the paralogous CCC subfamilies: the Na+ K+ 2Cl− cotransporter (NKCC1+2) and Na+ Cl− cotransporter (NCC), the K+–Cl− cotransporter (KCC1–4), the polyamine transporter CCC9, and the CCC-interacting protein CIP1 (6Gamba G. Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporter.Physiol. Rev. 2005; 85: 423-493Crossref PubMed Scopus (654) Google Scholar, 7Hartmann A.-M. Tesch D. Nothwang H.G. Bininda-Emonds O. Evolution of the cation chloride cotransporter family: ancient origins, gene losses, and subfunctionalization through duplication.J. Mol. Biol. Evol. 2014; 31: 434-447Crossref PubMed Scopus (49) Google Scholar). NKCCs and NCCs mediate the inward-directed transport of Na+, (K+), and Cl−, and KCCs mediate the outward-directed transport of K+ and Cl− (6Gamba G. Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporter.Physiol. Rev. 2005; 85: 423-493Crossref PubMed Scopus (654) Google Scholar). Both use Na+ and K+ gradients, which are built up by the Na+/K+ ATPase, to transport Cl− against its electrochemical gradient (6Gamba G. Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporter.Physiol. Rev. 2005; 85: 423-493Crossref PubMed Scopus (654) Google Scholar). CCC family members play an essential physiological role in transepithelial ion reabsorption and secretion, cell volume regulation, and Cl− homeostasis (6Gamba G. Molecular physiology and pathophysiology of electroneutral cation-chloride cotransporter.Physiol. Rev. 2005; 85: 423-493Crossref PubMed Scopus (654) Google Scholar, 8Payne J.A. Rivera C. Voipo J. Kaila K. 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To this important residues of both Cl−-binding sites were residues of This in the murine and showed to in human and a KCC2 transport The transport by of in as described A.-M. C. et in the large extracellular loop the cotransporters KCC2 and Biol. 2010; PDF PubMed Scopus Google Scholar, A.-M. C. Nothwang H.G. Structural changes in the extracellular loop 2 of the murine KCC2 potassium chloride cotransporter modulate ion Biol. PDF PubMed Scopus Google Scholar, J.A. of the for J. Physiol. PubMed Google Scholar). the transport to activity. of the of and The Cl1 residue with and with showed KCC2 These the of and in for Cl− coordination at of the site activity. were with or with mutations in the site were in for and were to the transport activity. The transport for in with with and with in and with in KCC2 transport The the of at including to in 2 with for to the of the and cell by were for the the for and of were the for and as is also in The human cell K+–Cl− transmembrane of site mutations in in to in to Open in a To the of the sites for KCC2 function, we the and in and in The mutation with KCC2 transport activity. The to and with showed KCC2 transport a crucial role in KCC2 Thus, Cl− in Cl2 is by of and in and of Tyr569 in that both Cl1 and Cl2 are for KCC2 transport of site mutations in in to in to Open in a we showed that an with an of a 3xHA tag in in conformational changes of and switches the site to an site to consist of and A.-M. C. Nothwang H.G. Structural changes in the extracellular loop 2 of the murine KCC2 potassium chloride cotransporter modulate ion Biol. PDF PubMed Scopus Google Scholar). To investigate, whether this extension of also an on the two Cl−-binding sites, we the for Cl1 and and for Cl2. The construct the same transport as and a of sites via A.-M. C. Nothwang H.G. Structural changes in the extracellular loop 2 of the murine KCC2 potassium chloride cotransporter modulate ion Biol. PDF PubMed Scopus Google Scholar). showed in with a KCC2 transport and The Cl1 mutation with and with and with KCC2 transport These are in with the analyses in KCC2WT and the of Cl− in Cl1 for KCC2 function. In addition, a with to the K+ coordination which is A.-M. C. Nothwang H.G. Structural changes in the extracellular loop 2 of the murine KCC2 potassium chloride cotransporter modulate ion Biol. PDF PubMed Scopus Google of site mutations in in to in to Open in a the with and with KCC2 transport and of to with These are in with the analyzed Cl−-binding sites in Cl2 for KCC2WT to the with KCC2 transport activity. Thus, the extension of the with the 3xHA tag affects of of and with Cl− in of site mutations in in to in to Open in a In the construct, we a and sites to the site of and A.-M. C. Nothwang H.G. Structural changes in the extracellular loop 2 of the murine KCC2 potassium chloride cotransporter modulate ion Biol. PDF PubMed Scopus Google Scholar). To the role of we the via and showed in to a KCC2 transport The with with and with KCC2 transport These the that the extension of with a 3xHA tag in conformational changes of the which switches the site to the site and of site mutations in in to in to Open in a To mutations in affect its transport we the and in showed in with a KCC2 transport The with and with showed transport with KCC2 Thus, is important for coordinating K+ at the site as as at the K+ showed that mutation of to in KCC2 transport A.-M. C. Nothwang H.G. Structural changes in the extracellular loop 2 of the murine KCC2 potassium chloride cotransporter modulate ion Biol. PDF PubMed Scopus Google Scholar). This is in with and its role in K+ coordination. The of may by a coordinating role of at of in and in to have already to reduce B. C. et structures of the human cation-chloride cotransporter PubMed Scopus (49) Google Scholar). The only residue that showed a is In mutation of to KCC2 in KCC2 activity. This that plays a crucial role in coordinating K+ at the K+ To that the of the tag to a of the K+ coordination site a of KCC2HA is of site mutations in in to in to Open in a structural analyses of CCCs by evolutionary highly conserved K+ and Cl− coordinating sites of the human cotransporter by PubMed Scopus Google Scholar, J. et al.The structural of and of neuronal cotransporters and Biol. PubMed Scopus Google Scholar, C. J. et and an of human Scopus Google Scholar, J. and transport of the and Mol. Biol. PubMed Scopus Google Scholar, of the cotransporter in Scopus Google Scholar, J. et and of the cotransporter PubMed Scopus Google Scholar, B. 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C. et structures of the human cation-chloride cotransporter PubMed Scopus (49) Google Scholar). This the question of whether both Cl− coordinating sites are essential for KCC2 function. of in that of Cl− in Cl2 reduces or abolishes the of and indicating that the Cl2 site is important for of the cotransporter in Scopus Google Scholar, J. et and of the cotransporter PubMed Scopus Google Scholar, B. C. et structures of the human cation-chloride cotransporter PubMed Scopus (49) Google Scholar, J. et of the human cotransporter Scopus Google Scholar). we demonstrated that mutation of residues coordinating Cl− in Cl1 and in or Cl2 in and Tyr569 in or KCC2 activity. In addition, in a role in K+ and Cl− in Cl1 J. et al.The structural of and of neuronal cotransporters and Biol. PubMed Scopus Google Scholar). of KCC2 indicating a crucial role in coordinating both A.-M. C. Nothwang H.G. 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