title: Rate and regulation of copper transport by human copper transporter 1 (hCTR1).
abstract: Human copper transporter 1 (hCTR1) is a homotrimer of a 190-amino acid monomer having three transmembrane domains believed to form a pore for copper permeation through the plasma membrane. The hCTR1-mediated copper transport mechanism is not well understood, nor has any measurement been made of the rate at which copper ions are transported by hCTR1. In this study, we estimated the rate of copper transport by the hCTR1 trimer in cultured cells using (64)Cu uptake assays and quantification of plasma membrane hCTR1. For endogenous hCTR1, we estimated a turnover number of about 10 ions/trimer/s. When overexpressed in HEK293 cells, a second transmembrane domain mutant of hCTR1 (H139R) had a 3-fold higher Km value and a 4-fold higher turnover number than WT. Truncations of the intracellular C-terminal tail and an AAA substitution of the putative metal-binding HCH C-terminal tripeptide (thought to be required for transport) also exhibited elevated transport rates and Km values when compared with WT hCTR1. Unlike WT hCTR1, H139R and the C-terminal mutants did not undergo regulatory endocytosis in elevated copper. hCTR1 mutants combining methionine substitutions that block transport (M150L,M154L) on the extracellular side of the pore and the high transport H139R or AAA intracellular side mutations exhibited the blocked transport of M150L,M154L, confirming that Cu(+) first interacts with the methionines during permeation. Our results show that hCTR1 elements on the intracellular side of the hCTR1 pore, including the carboxyl tail, are not essential for permeation, but serve to regulate the rate of copper entry.
authors: Department of Biochemistry and Molecular Genetics, University of Illinois College of Medicine, Chicago, IL 60607, USA.
authors: Edward B
authors: EB
authors: Maryon
authors: Shannon A
authors: SA
authors: Molloy
authors: Kristin
authors: K
authors: Ivy
authors: Huijun
authors: H
authors: Yu
authors: Jack H
authors: JH
authors: Kaplan
mesh_terms: D015153:Blotting, Western; D018938:Caco-2 Cells; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003300:Copper / Q000378:metabolism* / Q000493:pharmacokinetics; D003301:Copper Radioisotopes; D000079923:Copper Transporter 1; D004705:Endocytosis; D057809:HEK293 Cells; D006801:Humans; D017136:Ion Transport; D007700:Kinetics; D018613:Microscopy, Confocal; D009154:Mutation; D055503:Protein Multimerization
chemical_list: D027682:Cation Transport Proteins; D003301:Copper Radioisotopes; D000079923:Copper Transporter 1; C490079:SLC31A1 protein, human; D003300:Copper
keywords: Copper; Membrane Proteins; Membrane Transport; Metals; Transport Metals
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