Selective basolateral localization of overexpressed Na-K-ATPase β₁- and β₂- subunits is disrupted by butryate treatment of MDCK cells

Abstract

The exclusive basolateral localization of the Na-K-ATPase in kidney epithelium is a critical aspect of nephron function. It has been suggested that mislocalized delivery of the Na-K-ATPase to the apical surface in autosomal dominant polycystic kidney disease (ADPKD) is due to the inappropriate expression of an alternative isoform of the beta-subunit, the beta(2)-isoform. It has been reported that heterologous expression of this beta(2)-isoform in Madin-Darby canine kidney (MDCK) cells results in apical delivery of the Na-K-ATPase. We created a MDCK cell line containing a tetracycline-inducible promoter and expressed either myc-tagged beta(2)- or flag-tagged beta(1)-subunits to study the surface localization of these beta-subunit isoforms in polarized monolayers. We find that the beta(2)-isoform is targeted to the basolateral surface of the plasma membrane in a polarization pattern indistinguishable from the beta(1)-isoform. However, inclusion of butyrate in the growth medium leads to upregulation of overexpressed beta(1)- or beta(2)-subunits and to their appearance at the apical surface. The beta(2)-isoform expressed in MDCK cells does not assemble into alpha(1)beta(2) heterodimers with the endogenous alpha(1). Our findings demonstrate that expression of the beta(2)-isoform does not lead to apical localization of the Na-K-ATPase in MDCK cells and provides evidence for an unexpected effect of butyrate on the trafficking of Na pump subunits.

Human Copper Transporter hCTR1 Mediates Basolateral Uptake of Copper into Enterocytes

“…In these cells the exclusively basolateral expression of the Na,K-ATPase is unaffected, which implies that the trafficking pathways for the basolateral transport proteins, hCTR1 and Na,K-ATPase, are different. This conclusion is supported by our observation that overexpression of the basolateral ␤1-subunit of the Na,KATPase using this same promoter system does not cause any change in its localization (28).…”

“…The model also provides an explanation for why intestinal cells that selectively lack hCTR1 might be expected to still accumulate copper and yet not be able to pass it on to other tissues in the organism. The model also provides a function for the intracellular presence of hCTR1 recently reported in intestinal cells (21,22,28) and observed by us as sub-apical staining in mouse duodenal tissue.…”

“…8b). We have also previously shown that overexpression of the ␤1 subunit of the Na,K-ATPase in these same MDCK cells does not alter its exclusive basolateral targeting (28). The mechanistic basis of the loss of selective basolateral localization of overexpressed hCTR1 in MDCK cells is the subject of ongoing studies.…”

Acquisition of dietary copper: a role for anion transporters in intestinal apical copper uptake

“…Detection of overexpressed hCTR1 protein in total membranes (see Ref. 27 for the total membrane preparation) was analyzed by Western blot as described previously (53), using antibody to the FLAG-tag epitope at 1:1,000 dilution (no. F-3165, Sigma ) and anti-actin antibody (1:2,000; no.…”

β-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells

“…In this study, we inserted into the FRT site either Na-K-ATPase sheep ␤ 1-or rat ␤2-subunit with either a flag or Myc epitope tag on the carboxyl terminus. ␤1 with a flag tag (␤1flag), ␤1 with a Myc tag (␤1myc), or ␤2 with a Myc tag (␤2myc) were transfected into cells according to manufacturer's protocols using Lipofectamine 2000 (Invitrogen), as previously described (21). Another cell line containing both a Tet-regulatable ␤ 1flag subunit and a stable ␤1myc subunit was created by transfecting MDCK/␤1flag cells with pcDNA3.1 vector (Invitrogen) containing sheep ␤1-subunit with a carboxyl terminal Myc epitope tag (MDCK/␤1flag/␤1myc).…”