Yc. Qian et al., A MENKES P-TYPE ATPASE INVOLVED IN COPPER HOMEOSTASIS IN THE CENTRAL-NERVOUS-SYSTEM OF THE RAT, Molecular brain research, 48(1), 1997, pp. 60-66
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.