Publication Date

2013

Document Type

Thesis

Committee Members

Mauricio Difulvio (Advisor), Khalid Elased (Committee Member), Barbara Hull (Committee Member)

Degree Name

Master of Science (MS)

Abstract

The Na+K+2Cl- co-transporter-1 (NKCC1) effects the electroneutral movement of Na+, K+ and 2Cl- ions across plasma membranes. NKCC1 is considered a widely distributed, highly N-glycosylated ion transporter involved in the regulation of the intracellular chloride concentration ([Cl-]i), cell volume and salt secretion. NKCC1 locates in the plasma membrane of all cells studied so far, particularly on the basolateral side of most polarized cells. However, the majority of immunoreactive NKCC1 distributes in intracellular compartments of unknown nature whereas a small proportion of the transporter appears to be located in the plasma membrane. Here, we identified and characterized the molecular expression pattern of NKCC1 in the monkey fibroblast-like COS7 cell line and determined the impact of N-glycosylation on the cellular distribution of endogenous NKCC1 in these cells. We show that one of the two splice variants of NKCC1 i.e., NKCC1a is expressed in COS7 cells as a core/high mannose and complex N-glycosylated form in an approximate 4:1 ratio. Moreover, we demonstrate that NKCC1a locates in endoplasmic reticulum, medial-Golgi cisternae and recycling endosomes but minimally at the plasma membrane. We estimated that ~5-10 % of total NKCC1a expressed in COS7 cells reaches the plasma membrane in a functional state and in a comparable core/high-mannose to complex N-glycan proportion as the one observed in total extracts suggesting that N-glycosylation of the NKCC1a molecule is not a steady state signal for plasma membrane targeting of the transporter. Further, treatment of cells with tunicamycin, a potent inhibitor of the first step of N-glycan biosynthesis, results in endoplasmic reticulum retention and decreased total and plasma membrane located NKCC1a protein expression levels without affecting the ratio between core/high-mannose and complex N-glycosylated NKCC1a, indicating that the N-glycan nature of NKCC1a depends on its biosynthesis. Moreover, inhibition of N-glycan processing/maturation with kifunensine or swainsonine decreased basal complex N-glycosylated NKCC1a expression but increased basal expression levels of core/high-mannose N-glycosylated NKCC1a at the plasma membrane, suggesting that NKCC1a is delivered to this compartment independently of the N-glycan nature of the transporter. Altogether, our results are consistent with the conclusion that most of endogenous NKCC1a locates in defined intracellular compartments and that the N-glycan nature of the transporter does not dictate the trafficking of the transporter from one compartment to another, but rather reflects the biosynthetic pathway of the protein.

Page Count

124

Department or Program

Microbiology and Immunology

Year Degree Awarded

2013

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