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

Richa Singh,Mohamed Y Almiahoub,Shams Kursan,Tomás Garzón-Muvdi,Mauricio Di Fulvio,Mohammed M Almutairi,Francisco J Alvarez-Leefmans

doi:10.3389/fcell.2016.00150

Richa Singh, Mohamed Y Almiahoub + Show 5 more

Open Access

https://doi.org/10.3389/fcell.2016.00150

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Abstract

Na+K+2Cl− co-transporters (NKCCs) effect the electroneutral movement of Na+-K+ and 2Cl− ions across the plasma membrane of vertebrate cells. There are two known NKCC isoforms, NKCC1 (Slc12a2) and NKCC2 (Slc12a1). NKCC1 is a ubiquitously expressed transporter involved in cell volume regulation, Cl− homeostasis and epithelial salt secretion, whereas NKCC2 is abundantly expressed in kidney epithelial cells of the thick ascending loop of Henle, where it plays key roles in NaCl reabsorption and electrolyte homeostasis. Although NKCC1 and NKCC2 co-transport the same ions with identical stoichiometry, NKCC1 actively co-transports water whereas NKCC2 does not. There is growing evidence showing that NKCC2 is expressed outside the kidney, but its function in extra-renal tissues remains unknown. The present study shows molecular and functional evidence of endogenous NKCC2 expression in COS7 cells, a widely used mammalian cell model. Endogenous NKCC2 is primarily found in recycling endosomes, Golgi cisternae, Golgi-derived vesicles, and to a lesser extent in the endoplasmic reticulum. Unlike NKCC1, NKCC2 is minimally hybrid/complex N-glycosylated under basal conditions and yet it is trafficked to the plasma membrane region of hyper-osmotically challenged cells through mechanisms that require minimal complex N-glycosylation or functional Golgi cisternae. Control COS7 cells exposed to slightly hyperosmotic (~6.7%) solutions for 16 h were not shrunken, suggesting that either one or both NKCC1 and NKCC2 may participate in cell volume recovery. However, NKCC2 targeted to the plasma membrane region or transient over-expression of NKCC2 failed to rescue NKCC1 in COS7 cells where NKCC1 had been silenced. Further, COS7 cells in which NKCC1, but not NKCC2, was silenced exhibited reduced cell size compared to control cells. Altogether, these results suggest that NKCC2 does not participate in cell volume recovery and therefore, NKCC1 and NKCC2 are functionally different Na+K+2Cl− co-transporters.

Highlights

The Na+K+2Cl− co-transporters (NKCCs) are membrane proteins that carry out the electroneutral movement of Na+, K+, and 2Cl− ions across the plasma membrane
Using immunolabeling methods it was shown that COS7 express low but significant levels of NKCC2 (Alshahrani et al, 2015)
The results show that NKCC2 and Maackia amurensis lectin (MAL) do not co-localize further suggesting that plasma membrane localization of the co-transporter is minimal or undetectable under basal control conditions in COS7 cells (Figures 2G–L)

Summary

Introduction

The Na+K+2Cl− co-transporters (NKCCs) are membrane proteins that carry out the electroneutral movement of Na+, K+, and 2Cl− ions across the plasma membrane These symporters belong to the Slc12a family of solute carriers, which comprise at least eight homologous genes: Slc12a1 (NKCC2), Slc12a2 (NKCC1), Slc12a3 (Na+-Cl− co-transporter, NCC), Slc12a47 (K+-Cl− co-transporters, KCC1-4), and Slc12a9 (Hartmann et al, 2014). 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). In this location, NKCC2 plays a key role in salt reabsorption and urine concentration (Castrop and Schiessl, 2014). NKCC2 expression has been reported in enteric neurons (Xue et al, 2009), gastric, intestinal, endolymphatic sac, and olfactory epithelia (Akiyama et al, 2007, 2010; Nickell et al, 2007; Nishimura et al, 2009; Xue et al, 2009; Zhu et al, 2011; Ji et al, 2012), starburst amacrine cells (Gavrikov et al, 2006), chondrocytes (Bush et al, 2010), and endocrine/neuroendocrine cells including insulin secreting β-cells of the pancreas (Corless et al, 2006; Bensellam et al, 2009; Ghanaat-Pour and Sjöholm, 2009; Alshahrani et al, 2012; Alshahrani and Di Fulvio, 2012) and vasopressinergic/oxytocinergic neurons of the supraoptic and paraventricular nuclei (Hindmarch et al, 2006; Konopacka et al, 2015)

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