RGD Reference Report - Identification of a functionally important conformation-sensitive region of the secretory Na+-K+-2Cl- cotransporter (NKCC1). - Rat Genome Database

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Identification of a functionally important conformation-sensitive region of the secretory Na+-K+-2Cl- cotransporter (NKCC1).

Authors: Dehaye, JP  Nagy, A  Premkumar, A  Turner, RJ 
Citation: Dehaye JP, etal., J Biol Chem. 2003 Apr 4;278(14):11811-7. Epub 2003 Jan 29.
RGD ID: 1580435
Pubmed: PMID:12556450   (View Abstract at PubMed)
DOI: DOI:10.1074/jbc.M213148200   (Journal Full-text)

The secretory Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is a member of a small gene family of electroneutral salt transporters that play essential roles in salt and water homeostasis in many mammalian tissues. We have identified a highly conserved residue (Ala-483) in the sixth membrane-spanning segment of rat NKCC1 that when mutated to cysteine renders the transporter sensitive to inhibition by the sulfhydryl reagents 2-aminoethyl methanethiosulfonate (MTSEA) and 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The mutation of Ala-483 to cysteine (A483C) results in little or no change in the affinities of NKCC1 for substrate ions but produces a 6-fold increase in sensitivity to the inhibitor bumetanide, suggesting a specific modification of the bumetanide binding site. When residues surrounding Ala-483 were mutated to cysteine, only I484C was sensitive to inhibition by MTSEA and MTSET. Surprisingly I484C showed increased transport activity in the presence of low concentrations of mercury (1-10 microm), whereas A483C showed inhibition. The inhibition of A483C by MTSEA was unaffected by the presence or absence of sodium and potassium but required the presence of extracellular chloride. Taken together, our results indicate that Ala-483 lies at or near an important functional site of NKCC1 and that the exposure of this site to the extracellular medium is dependent on the conformation of the transporter. Specifically, our results indicate that the cysteine introduced at residue 483 is only available for interaction with MTSEA when chloride is bound to NKCC1 at the extracellular surface.



Gene Ontology Annotations    Click to see Annotation Detail View

Biological Process

  
Object SymbolSpeciesTermQualifierEvidenceWithNotesSourceOriginal Reference(s)
Slc12a2Ratchloride transport  IMP  RGD 
Slc12a2Ratpotassium ion transport  IMP  RGD 
Slc12a2Ratsodium ion transport  IMP  RGD 

Molecular Function

  
Object SymbolSpeciesTermQualifierEvidenceWithNotesSourceOriginal Reference(s)
Slc12a2Ratsodium:potassium:chloride symporter activity  IMP  RGD 

Objects Annotated

Genes (Rattus norvegicus)
Slc12a2  (solute carrier family 12 member 2)


Additional Information