Fig. 2.
Copper chelators induced steady-state NMDAR current. (A) Chelating copper with bathocuproine disulfonate (BCS) reversibly induced steady-state NMDAR current in rat hippocampal neurons. (B) Representative time course of development of the steady-state current after addition of BCS. (C) Mean ± SEM steady-state current as a percentage of peak. There was minimal steady-state current in the absence of copper chelators (white bars), whereas copper chelators induced a substantial steady-state component. (D) Specificity of BCS was determined by replacing metal ions. Addition of 2 μM copper to 10 μM BCS had no effect as the chelator remained substantially in excess. In contrast, addition of 2 μM zinc completely abolished the steady-state current, indicating that BCS does not appreciably bind zinc at the concentrations used. Adding excess copper to BCS predictably abolished the steady-state current. (E) Effect of BCS was not additive with that of Aβ1–42. (Inset) Representative traces scaled to normalized peak currents. (F) Disrupting copper homeostasis by exposure of neurons to BCS (10 μM) induced significant neuronal death as measured by TUNEL labeling. Aβ1–42 was equally toxic, but scrambled Aβ1–42 (subjected to the same oligomerization procedure as normal sequence peptide) was not. NMDAR block with 5,7-dichlorokynurenic acid (DCKA, 100 μM) or replenishment with CuSO4 (4 μM) protected neurons from injury. Neurons exposed to NMDA and d-serine (both 500 μM) served as positive controls. Values are mean ± SEM. *P < 10−5; #P > 0.95 vs. control.