Endocytic recycling of Na⁺–K⁺–Cl⁻ cotransporter type 2: importance of exon 4

Abstract

Key points Na+–K+–Cl− cotransporter type 2 (NKCC2) is a 27‐exon membrane protein that is expressed in the thick ascending limb (TAL) of Henle where it is involved in reabsorption of the ultrafiltered NaCl load. It comes as three splice variants that are identical to each other except for the residue composition of exon 4 and that differ in their transport characteristics, functional roles and distributions along the TAL. In this report, it is shown that the variants also differ in their trafficking properties and that two residues in exon 4 play a key role in this regard. One of these residues was also shown to sustain carrier internalization. Through these results, a novel function for the alternatively spliced exon of NKCC2 has been identified and a domain that is involved in carrier trafficking has been uncovered for the first time in a cation–Cl− cotransporter family member. Abstract 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.

Molecular characteristics and physiological roles of Na+–K+–Cl− cotransporter 2

“…In simulation analyses Chew et al further showed that the CL1 domain of one monomer could interact with the C-terminus of the opposite monomer. This finding was of special interest in view of the role that the TMD2-CL1 domain of NKCC2 was found to play (Gagnon et al, 2004(Gagnon et al, , 2005(Gagnon et al, , 2002Gimenez et al, 2002;Marcoux et al, 2019aMarcoux et al, , 2019bPlata et al, 2002). Similar analyses also showed that the ion-binding sites were contained within a solvent-accessible vestibule that was delineated by TMD1, TMD3, TMD6, and TMD8.…”

“…The apparent ion affinity (K m ) and maximal transport capacity (V max ) of the NKCC2 splice variants have been determined by at least three research groups in the oocyte expression system (Gimenez et al, 2002;Marcoux et al, 2019aMarcoux et al, , 2019bPlata, Meade, Vazquez, Hebert, & Gamba, 2002). Among the experiments carried out by these research groups, the NKCC2 analyzed were not all from the same species and the conditions used differed to some degree.…”

“…Through subsequent studies, a mutagenic approach allowed Marcoux et al (2019a) to identify variants residues in CL1 (at Positions 230 and 238) that endowed each of the variants with unique trafficking and ion transport characteristics. From these studies, it was concluded that CL1 could be part of a key regulatory motif that was not only involved in carrier trafficking but that also contributed to the differential properties of Na + -K + -Cl − cotransport along the TALH.…”

Phospho‐regulation, nucleotide binding and ion access control in potassium‐chloride cotransporters

“…Rb + flux assays Synthetic genes for KCC1 and KCC3 variants with N-terminal HAtag were cloned into pPOL vector for in vitro transcription (Marcoux et al, 2019). Defolliculated stage V-VI Xenopus laevis oocytes were microinjected with 50 ng cRNA and maintained in Barth medium for 3 days at 18°C in the presence of 1.5 mM furosemide.…”