Human Copper Transporter hCTR1 Mediates Basolateral Uptake of Copper into Enterocytes

Abstract

Copper is essential for human growth and survival. Enterocytes mediate the absorption of dietary copper from the intestinal lumen into blood as well as utilizing copper for their biosynthetic needs. Currently, the pathways for copper entry into enterocytes remain poorly understood. We demonstrate that the basolateral copper uptake into intestinal cells greatly exceeds the apical uptake. The basolateral but not apical transport is mediated by the high affinity copper transporter hCTR1. This unanticipated conclusion is supported by cell surface biotinylation and confocal microscopy of endogenous hCTR1 in Caco2 cells as well as copper influx measurements that show saturable high affinity uptake at the basolateral but not the apical membrane. Basolateral localization of hCTR1 and polarized copper uptake are also conserved in T84 cells, models for intestinal crypt cells. The lateral localization of hCTR1 seen in intestinal cell lines is recapitulated in immunohistochemical staining of mouse intestinal sections. Biochemical and functional assays reveal the basolateral localization of hCTR1 also in renal MadinDarby canine kidney cells and opossum kidney cells. Overexpression of hCTR1 in Madin-Darby canine kidney cells results in both apical and basolateral delivery of the overexpressed protein and greatly enhanced copper uptake at both cell surfaces. We propose a model of intestinal copper uptake in which basolateral hCTR1 plays a key role in the physiologically important delivery of copper from blood to intracellular proteins, whereas its role in the initial apical uptake of dietary copper is indirect.

Rate and Regulation of Copper Transport by Human Copper Transporter 1 (hCTR1)

“…64 Cu uptake assays in Caco-2 cells were performed in 6-well Transwell plates. Caco-2 cells were plated on 24-mm polyester membrane Transwell inserts and cultured as described previously (30). Copper uptake measurements were performed at 2.5 M CuCl 2 on either apical or basolateral membrane of polarized Caco-2 cells as described previously (30) Duplicate Transwell culture plates were used for protein determination.…”

“…All of the biotinylation procedures were performed at 4°C. Caco-2 cells were grown in Transwell culture plates until polarized, and either the apical or the basolateral membrane was biotinylated (30,31).…”

“…HEK293 cells were subcultured every 3-5 days. Caco-2 cells were grown on Transwell culture plates as described previously (30).…”

“…Cells were incubated in standard DMEM with 10% FBS and further supplemented with 2 M CuCl 2 containing trace amounts of 64 Cu. In this medium, under these conditions, 70 -85% copper uptake occurs via hCTR1 (11,12,30). We performed the assay in 12-well culture plates having confluent monolayers of cells, using 3-8 replicate wells for each condition.…”

Posttranslational regulation of copper transporters

“…3) [38,39,79]. The subcellular localization of hCTR1 is regulated by extracellular copper concentrations.…”