DEFECTIVE INTRACELLULAR COPPER TRANSLOCATION IN MENKES KINKY HAIR SYNDROME

Abstract

Menkes kinky hair syndrome is an x-linked neurode-generative disorder causing tissue-specific increases in copper (Cu) and metallothionein (MT) concentrations. Previous work has shown that MT gene regulation may be normal in Menkes. However, abnormalities in Cu retention and utilization by mutant cells suggest that the basic defect may reside in the transfer of Cu across cell compartments. We tested this hypothesis in Menkes fibroblasts by examining the distribution of Cu and zinc (Zn) in crude particulate and cytosolic fractions (pellet and supernatant of a 100,000 g-min spin). In control cells, the Cu content of particulate fractions incrementally rose up to 2 mcg/mg cell protein as cytosolic Cu content was increased (up to 1.4 mcg/mg protein) by growth conditions. In contrast, in Menkes cells, over a range of cytosolic Cu concentrations indistinguishable from that in the control cells (P>0.10, Kolmogorov-Smirnov test), particulate Cu content remained significantly lower than in controls (≤0.3 mcg/mg protein, P<0.001, extended Mantel-Haenszel test). A similar difference in dose-response was observed when 64-Cu accumulation was assessed. At overlapping cytosolic 64-Cu concentrations, 64-Cu appeared in control particulate fractions in significantly higher concentrations than in Menkes cells. In contrast, particulate-cytosol distributions in 65-Zn accumulation studies were identical in Menkes and control. We conclude that a major consequence of the Menkes mutation is a specific failure of Cu translocation across a cell compartment.