Copper efflux from murine microvascular cells requires expression of the menkes disease Cu-ATPase.
Previously, we showed that the transport of Cu by PC12 pheochromocytoma cells and C6 glioma cells correlated with the expression of a Cu-transporting ATPase (Atp7a) that has been linked to Menkes disease. Here, we show that cerebrovascular endothelial (CVE) cells that comprise the blood-brain barrier (BBB) also express the gene for the Cu-ATPase. By using reverse transcription-polymerase chain reaction (RT-PCR) and primers designed from mouse Atp7a cDNA, we amplified a 925-bp and a 760-bp cDNA fragment from two extreme regions of Atp7a mRNA from murine CVE cells; 777 bp of the 925-bp fragment and 677 bp of the 760-bp fragment had a 99.7 and 100% sequence homology, respectively, with mouse Atp7a cDNA. The 777-bp sequences covered the heavy metal binding (Hmb) domain and the 677-bp fragment coded for residues at the -COOH terminus of Atp7a. A functional analysis showed that Cu efflux was blocked by the sulfhydryl reagent p-chloromercuribenzoate (p-CMB), a potential inhibitor of Atp7a function. This study provides strong evidence that a Cu-ATPase in the BBB controls the penetration of Cu into the brain and that lesions to the Cu-ATPase in CVE cells are a primary cause of low brain Cu levels in Menkes disease.
Departments of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.
Y
Y
Qian
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Tiffany-Castiglioni
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Welsh
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ED
Harris
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D001812:Blood-Brain Barrier; D001921:Brain / Q000098:blood supply*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D027682:Cation Transport Proteins; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D018076:DNA, Complementary / Q000737:chemistry; D004730:Endothelium, Vascular / Q000201:enzymology*; D015870:Gene Expression; D007706:Menkes Kinky Hair Syndrome / Q000201:enzymology*; D051379:Mice; D008833:Microcirculation / Q000201:enzymology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase; D011993:Recombinant Fusion Proteins; D017385:Sequence Homology
C486201:Atp7a protein, mouse; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D003300:Copper; D012194:RNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases