Impact of Hybrid and Complex N-Glycans on Cell Surface Targeting of the Endogenous Chloride Cotransporter Slc12a2.
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.
Department of Biological Sciences, Boonshoft School of Medicine, Wright State University, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA.
Richa
R
Singh
Department of Pharmacology & Toxicology, Boonshoft School of Medicine, Wright State University, 3640 Colonel Glenn Highway, 216 HSB, Dayton, OH 45435, USA.
Mohammed Mashari
MM
Almutairi
Department of Pharmacology & Toxicology, Boonshoft School of Medicine, Wright State University, 3640 Colonel Glenn Highway, 216 HSB, Dayton, OH 45435, USA.
Romario
R
Pacheco-Andrade
Department of Pharmacology & Toxicology, Boonshoft School of Medicine, Wright State University, 3640 Colonel Glenn Highway, 216 HSB, Dayton, OH 45435, USA.
Mohamed Y Mahmoud
MY
Almiahuob
Department of Pharmacology & Toxicology, Boonshoft School of Medicine, Wright State University, 3640 Colonel Glenn Highway, 216 HSB, Dayton, OH 45435, USA.
Mauricio
M
Di Fulvio