Endocytic recycling of Na+ -K+ -Cl- cotransporter type 2: importance of exon 4.

Na+ -K+ -Cl- cotransporter type 2 (NKCC2) is a 12-transmembrane (TM) domain cell surface glycoprotein that is expressed in the thick ascending limb (TAL) of Henle and stimulated during cell shrinkage. It comes as three splice variants (A, B and F) that are identical to each other except for TM2 and the following connecting segment (CS2). Yet, these variants do not share the same localization, transport characteristics and physiological roles along the TAL. We have recently found that while cell shrinkage could exert its activating effect by increasing NKCC2 expression at the cell surface, the variants also responded differentially to this stimulus. In the current work, a mutagenic approach was exploited to determine whether CS2 could play a role in carrier trafficking and identify the residues potentially involved. We found that when the residue of position 238 in NKCC2A (F) and NKCC2B (Y) was replaced by the corresponding residue in NKCC2F (V), carrier activity increased by over 3-fold and endocytosis decreased concomitantly. We also found that when the residue of position 230 in NKCC2F (M) was replaced by the one in NKCC2B (T), carrier activity and affinity for ions both increased substantially whereas expression at the membrane decreased. Taken together, these results suggest that CS2 is involved in carrier trafficking and that two of its residues, those of positions 238 and 230, are part of an internalization motif. They also indicate that the divergent residue of position 230 plays the dual role of specifying ion affinity and sustaining carrier internalization.