A Menkes P-type ATPase involved in copper homeostasis in the central nervous system of the rat.
We previously reported that copper efflux from C6 rat glioma cells was blocked by a brief exposure to sulfhydryl reagents p-chloromercuribenzoate (PCMB) and iodoacetamide as well as dicyclohexylcarbodiimide, suggesting the possible involvement of a Cu-transporting ATPase in the efflux mechanism. In this report, we show that copper efflux from PC12 cells, a neuron-like cell line established from rat adrenal pheochromocytoma, is also inhibited by PCMB exposure. Furthermore, we show that both C6 and PC12 cells express a homolog of the Menkes gene (MNK) as detected by RT-PCR with primers designed from a mouse cDNA and confirmed by sequence analysis of the amplified product. An expected 760-bp fragment representing the transduction and phosphorylation domains and a 925-bp fragment encoding the heavy metal-binding domain of Atp7a were amplified from a RNA extract of C6 and PC12 cells. Sequence data revealed that 690 bp of the 760-bp fragment from C6 cells were an identical match to a similar fragment from PC12 cells. Both fragments encoded a 229 amino-acid polypeptide that had a 98.7% sequence homology to mouse Atp7a. In addition, 880 bp from the 925-bp fragment of the two cell lines were identical and encoded a 293 amino-acid polypeptide with 94.5% sequence homology to mouse Atp7a. These data establish that a Menkes-type Cu-transporting ATPase is expressed in rat C6 and PC12 cells and strongly support the hypothesis that both neurons and glia are involved in maintaining Cu homeostasis in the central nervous system.
Qian
Y
Y
Department of Biochemistry and Biophysics, Texas A&M, College Station 77843, USA.
Tiffany-Castiglioni
E
E
Harris
E D
ED
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000310:Adrenal Gland Neoplasms; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D027682:Cation Transport Proteins; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D017931:DNA Primers; D018076:DNA, Complementary; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005910:Glioma; D006706:Homeostasis; D006801:Humans; D007460:Iodoacetamide / Q000494:pharmacology; D007706:Menkes Kinky Hair Syndrome / Q000235:genetics; D051379:Mice; D008959:Models, Neurological; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D016716:PC12 Cells; D010673:Pheochromocytoma; D016133:Polymerase Chain Reaction; D051381:Rats; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D020245:p-Chloromercuribenzoic Acid
C486201:Atp7a protein, mouse; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D002729:Chloromercuribenzoates; D017931:DNA Primers; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D013439:Sulfhydryl Reagents; D004024:Dicyclohexylcarbodiimide; D020245:p-Chloromercuribenzoic Acid; D003300:Copper; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; C486202:Atp7a protein, rat; D000073840:Copper-Transporting ATPases; D007460:Iodoacetamide