A Highly Conserved Motif at the COOH Terminus Dictates Endoplasmic Reticulum Exit and Cell Surface Expression of NKCC2

摘要: Mutations in the apically located Na+-K+-2Cl− co-transporter, NKCC2, lead to type I Bartter syndrome, a life-threatening kidney disorder, yet mechanisms underlying regulation of mutated NKCC2 proteins renal cells have not been investigated. Here, we identified trihydrophobic motif distal COOH terminus that was required for endoplasmic reticulum (ER) exit and surface expression co-transporter. Indeed, microscopic confocal imaging showed naturally occurring mutation depriving its COOH-terminal region results absence cell expression. Biotinylation assays revealed lack associated with abolition mature complex-glycosylated NKCC2. Pulse-chase analysis demonstrated protein caused by reduced synthesis or increased rates degradation mutant co-transporters. Co-immunolocalization experiments these mutants co-localized ER marker protein-disulfide isomerase, demonstrating they are retained ER. Cell treatment proteasome lysosome inhibitors failed restore loss further eliminating possibility co-transporters were processed Golgi apparatus. Serial truncation terminus, followed site-directed mutagenesis, hydrophobic residues 1081LLV1083 as an signal necessary maturation Mutation AAA within context full-length prevented independently system. This is highly conserved tails all members cation-chloride co-transporter family, thus may function common mediating their transport from surface. Taken together, data consistent model whereby premature terminations interfere LLV compromise delivery through defective trafficking.