Perinuclear localization of Na-K-Cl-cotransporter protein after human cytomegalovirus infection

Abstract

previously reported that Na-K-Cl-cotransporter (NKCC) function and microsomal protein expression are both dramatically reduced late in human cytomegalovirus (HCMV) infection of a human fibroblast cell line (MRC-5). We now report DNA microarray data showing that no significant HCMV-dependent NKCC gene repression can be detected 30 h postexposure (PE) to the virus. Consequently, we used plasma membrane biotinylation and subsequent subcellular fractionation in combination with semiquantitative immunoblotting and confocal microscopy to investigate the possibility that intracellular redistribution of the NKCC protein after HCMV infection could be a cause of the HCMV-induced loss of NKCC ion transport function. Our results show that the lifetime of plasmalemmal NKCC protein in quiescent, uninfected MRC-5 cells is ϳ48 h, and Ͻ20% of the total expressed NKCC protein are in the plasma membrane. The remainder (ϳ80%) was detected as diffusely distributed, small punctate structures in the cytoplasm. Following HCMV infection: 1) NKCC protein expression in the plasmalemma was sharply reduced (ϳ75%) within 24 h PE and thereafter continued to slowly decrease; 2) total cellular NKCC protein content remained unchanged or slightly increased during the course of the viral infection; and 3) HCMV infection caused NKCC protein to accumulate in the perinuclear region late in the HCMV infection (72 h PE). Thus our results imply that, in the process of productive HCMV infection, NKCC protein continues to be synthesized, but, instead of being delivered to the plasma membrane, it is clustered in a large, detergentsoluble perinuclear structure.sodium-potassium-chloride-cotransporter; human fibroblast cell line; perinuclear accumulation

A key role for membrane transporter NKCC1 in mediating chondrocyte volume increase in the mammalian growth plate

“…(21) Similar differences in NKCC distribution between PZCs and HZCs are also observed with human cytomegalovirus infection of a fibroblast cell line. (22) Infection inhibits translocation of NKCC to the plasma membrane, resulting in similar perinuclear distribution as shown here, with abolition of cell NKCC activity.…”

Functional Expression of the Na-K-2Cl Cotransporter NKCC2 in Mammalian Cells Fails to Confirm the Dominant-negative Effect of the AF Splice Variant

“…3 B ) of the cotransporter appeared in the biotinylated fraction. As larger sample sizes and longer exposure times were required to detect the transporters in the biotinylated compared with whole cell samples it appears that only a small fraction of total NKCC1 or fNKCC2 is in the surface membrane and can be biotinylated as suggested previously (31, 32). …”

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

“…Consistent with the negligible plasma membrane expression is the finding that integrin β2, a plasma membrane marker did not co-localize with NKCC2 (Figures 2A–F), and the co-transporter did not co-localize in lectin-labeled cells (Figures 2G–L). Further, NKCC2 and NKCC1, a co-transporter that localizes to the plasma membrane and in defined intracellular organelles (Maglova et al, 2004; Singh et al, 2015) share some but not all cellular compartments (Figures 2D–F). These results demonstrate that endogenous NKCC2 in COS7 cells is mostly expressed in intracellular compartments.…”