Impact of Hybrid and Complex N-Glycans on Cell Surface Targeting of the Endogenous Chloride CotransporterSlc12a2

Abstract

The Na+K+2Cl− cotransporter-1 (Slc12a2, NKCC1) is widely distributed and involved in cell volume/ion regulation. Functional NKCC1 locates in the plasma membrane of all cells studied, particularly in the basolateral membrane of most polarized cells. Although the mechanisms involved in plasma membrane sorting of NKCC1 are poorly understood, it is assumed that N-glycosylation is necessary. Here, we characterize expression, N-glycosylation, and distribution of NKCC1 in COS7 cells. We show that ~25% of NKCC1 is complex N-glycosylated whereas the rest of it corresponds to core/high-mannose and hybrid-type N-glycosylated forms. Further, ~10% of NKCC1 reaches the plasma membrane, mostly as core/high-mannose type, whereas ~90% of NKCC1 is distributed in defined intracellular compartments. In addition, inhibition of the first step of N-glycan biosynthesis with tunicamycin decreases total and plasma membrane located NKCC1 resulting in almost undetectable cotransport function. Moreover, inhibition of N-glycan maturation with swainsonine or kifunensine increased core/hybrid-type NKCC1 expression but eliminated plasma membrane complex N-glycosylated NKCC1 and transport function. Together, these results suggest that (i) NKCC1 is delivered to the plasma membrane of COS7 cells independently of its N-glycan nature, (ii) most of NKCC1 in the plasma membrane is core/hybrid-type N-glycosylated, and (iii) the minimal proportion of complex N-glycosylated NKCC1 is functionally active.

Plasma Membrane Targeting of Endogenous NKCC2 in COS7 Cells Bypasses Functional Golgi Cisternae and Complex N-Glycosylation

“…Lentiviral vectors expressing mCherry-tagged hNKCC2A WT were from GeneCopoeia (Rockville, MD). To silence endogenous NKCC1 or NKCC2 expression, COS7 cells were stably transfected with human lentiviral constructs encoding green fluorescent protein (GFP) and shRNAs against the 4th exon of hNKCC1, as previously described (Alshahrani et al, 2015; Singh et al, 2015) or against the 20th exon of hNKCC2. As control, we used constructs lacking shRNA sequences.…”

“…First-strand cDNA synthesis from total RNA and gene-specific PCR was done as described in detail elsewhere (Alshahrani et al, 2012; Grobe et al, 2015; Singh et al, 2015). Control RT-PCR reactions were performed using glyceraldehyde phosphate dehydrogenase (GAPDH, NM_002046) primers: GAPDH-555 sense: GTG AAG GTC GGA GTC AAC GGA TTT, GAPDH-555 antisense: CAC AGT CTT CTG GGT GGC AGT GAT.…”

“…Fifty to one hundred micrograms of total protein from COS7 cells were subjected to SDS-PAGE (pre-casted gels 4–20% Pierce, Thermo Scientific, Rockford, IL), transferred to PDVF membranes and blotted as described in detail elsewhere (Singh et al, 2015). β-actin expression was used as internal loading control.…”

“…Molecular and/or functional expression of NKCC1 and its spliced variants has been demonstrated in a variety of primary cells types, including cell lines (Alshahrani et al, 2012; Mao et al, 2012; Markadieu and Delpire, 2014; Singh et al, 2015). In contrast, the main products of the Slc12a1 gene (NKCC2) i.e., NKCC2A, NKCC2B, and NKCC2F, have long been considered to be exclusive to the apical membrane of the tubular cells of the thick ascending loop of Henle (TALH).…”

“…Since targeting of endogenous NKCC1 to the plasma membrane is independent of hybrid/complex N-glycosylation (Singh et al, 2015) and genetic deletion of NKCC1 in some cells results in permanent cell shrinkage (Crum et al, 2012), we hypothesized that NKCC2 expression increases in cells subjected to sustained osmotic shrinkage by mechanisms that do not require the classic secretory pathway. In the present report, we confirm and extend previous results by demonstrating that: (i) one splice variant of NKCC2, NKCC2A, is produced in COS7, (ii) NKCC2 is natively expressed in COS7 cells at relatively low levels, (iii) NKCC2 and NKCC1 co-localize to some but not all cellular compartments, (iv) under basal conditions endogenous NKCC2 localizes to the endoplasmic reticulum (ER), cis/medial Golgi cisternae, pericentriolar/microtubule organizing center, and endosomal compartments but not to lysosomes or the plasma membrane region, (v) NKCC2 expression and plasma membrane region localization increase in response to sustained exposure to hyperosmotic solutions and (vi) complex N-glycosylation or functional Golgi stacks are not required for NKCC2 targeting to the plasma membrane region.…”