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-chloromercuriben zoate (PCMB) and iodoacetamide as well as dicyclohexylcarbodiimide, su ggesting the possible involvement of a Cu-transporting ATPase in the e fflux mechanism. In this report, we show that copper efflux from PC12 cells, a neuron-like cell line established from rat adrenal pheochromo cytoma, is also inhibited by PCMB exposure. Futhermore, we show that b oth C6 and PC12 cells express a homolog of the Menkes gene (MNK) as de tected by RT-PCR with primers designed from a mouse cDNA and confirmed by sequence analysis of the amplified product. An expected 760-bp fra gment 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 revea led 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 mous e 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 Menke s-type Cu-transporting ATPase is expressed in rat C6 and PC12 cells an d strongly support the hypothesis that both neurons and glia are invol ved in maintaining Cu homeostasis in the central nervous system.