The multidrug resistance (mdr1) gene product functions as an ATP channel.

Abstract

The multidrug resistance (mdrl) gene product, P-glycoprotein, is responsible for the ATP-dependent extrusion of a variety of compounds, including chemotherapeutic drugs, from cells. The data presented here show that cells with increased levels of the P-glycoprotein release ATP to the medium in proportion to the concentration of the protein in their plasma membrane. Furthermore, measurements of whole-cell and single-channel currents with patch-clamp electrodes indicate that the P-glycoprotein serves as an ATPconducting channel in the plasma membrane. These findings suggest an unusual role for the P-glycoprotein.Multidrug resistance in tumor cells is mediated by increased expression of the product of the mdrl gene (MDRI, now PGYJ, in human gene nomenclature), the P-glycoprotein (1-5). Its primary structure (6, 7) is similar to that of various transporters of ions, amino acids, peptides, or proteins in bacterial, yeast, and animal cells, which share ATP-binding motifs (8). The P-glycoprotein also resembles structurally the mitochondrial ATP/ADP exchanger (9, 10).The hydrolysis of ATP apparently provides the energy for drug extrusion from the cell (11-19). However, the mechanism of the transport process is not known for any of the transporters of the ATP-binding cassette (ABC) superfamily, including one reconstituted in artificial vesicles (20).In this paper we-show that P-glycoprotein in plasma membranes serves as a channel for steady ATP release from the cell. The possible physiological significance ofthis export will be briefly discussed. MATERIALS AND METHODSCell Lines. Chinese hamster ovary (CHO) cells, designated AUX B1, and P-glycoprotein-overexpressing drug-resistant cells, CHRC5 and CHRB30, were provided by Victor Ling (Ontario Cancer Institute, Toronto) (21). The human lung tumor cell line SW-1573 mutants that express the P-glycoprotein in different amounts were obtained by culturing the cells with adriamycin at 5 or 7 ,ug/ml, yielding SW10 or SWadria,.Drug-sensitive CHO cells (LR73) (25) transfected with multiple copies of murine mdrl cDNA (EX4N), or transfected with the murine mdrl cDNA in which the codons for lysines 432 and 1074 had been replaced by those for arginine residues to modify the two nucleotide-binding sites (88-8), were obtained from Philippe Gros (McGill University, Montreal).Immunofluorescence. P-glycoprotein concentration on cell surfaces was measured by staining with fluorescent mouse monoclonal antibodies directed to surface epitopes of the protein, followed by analysis on a FACScan (Becton Dickinson) using LYSYS software. The hamster cell lines were stained with antibody 4A6B3/10 to the hamster P-glycoprotein (26), and the human cell lines were stained with antibody 4E3 to the human P-glycoprotein (R.J.A., unpublished results).ATP measurements. (i) HPLC. Nucleotides, in the Hanks buffer bathing the cells, were separated by HPLC (Waters) and quantitated as described by Fellenz and Gerweck (27).(ii) Luminometry. After stable baseline measurements were obtained from 1 ml of Hanks...

The role of purinergic signaling in the liver and in transplantation: effects of extracellular nucleotides on hepatic graft vascular injury, rejection and metabolism

“…Such pathways include exocytosis of ATP/UTP-containing vesicles, facilitated diffusion via connexin-43 hemichannels, by putative ABC transporters or potentially by poorly understood electrodiffusional movements through ATP/nucleotide channels (2,(15)(16)(17)(18)(19)(20)(21). Under pathophysiological conditions, the release of nucleotides and the expression of purinergic receptors is increased markedly in injured or stressed cells (17).…”

Secretion of IL-2 and IFN-γ, But Not IL-4, by Antigen-Specific T Cells Requires Extracellular ATP

“…Extracellular ATP does not cross the cell membrane, but rather mediates its biological actions through interaction with specific transmembrane proteins on the cell surface (1)(2)(3). Several proteins on the cell surface have been described that are capable of binding extracellular ATP and other nucleotides.…”

Function and Regulation of Pannexin 1 Channels in the Vascular Endothelium

“…In agreement with these findings, whole-cell and inside-out patch clamp recordings of cells transfected with CFTR revealed ATP currents that were dependent on cAMP and PKA activation, which were absent in cells lacking the ABC transporter [89]. Related studies show ATP currents in cells expressing the multiple drug resistance gene product P-glycoprotein, another member of the ABC transporter family, further supporting a functional role for this family of proteins in the release of ATP [88]. While the major focus of ATP release from CFTR has been in epithelial cell physiology, this transporter has been identified in vascular smooth muscle cells [95], endothelial cells [96] and circulating erythrocytes [28] and platelets [97] providing a potential conduit for ATP release into from these cells.…”

“…Each member of an ABC transporter has two conserved intracellular ATP binding domains that bind and hydrolyze ATP. Three of these proteins, the cystic fibrosis transmembrane conductance regulator (CFTR), the multidrug resistance gene product mdr (also known as P-glycoprotein) and the sulfonylurea receptor (SUR) have been suggested to not only utilize ATP as an energy source for active transport, but to physically transport the purine nucleotide out of the cell for autocrine/paracrine purinergic signaling [88][89][90]. With the search for a channel or transporter that is responsible for ATP release in a multitude of cell types, the ABC transporters have become a potential candidate to fill this role.…”