Abstract
Although the tricyclic antidepressant amitriptyline (ATL) is widely used in the clinic, the mechanism underlying its high therapeutic efficacy against neuropathic pain remains unclear. NMDA receptors (NMDARs) represent a target for ATL and are involved in sensitization of neuropathic pain. Here we describe two actions of ATL on NMDARs: 1) enhancement of Ca(2+)-dependent desensitization and 2) trapping channel block. Inhibition of NMDARs by ATL was found to be dependent upon external Ca(2+) concentration ([Ca(2+)]) in a voltage-independent manner, with an IC(50) of 0.72 μM in 4 mM [Ca(2+)]. The ATL IC(50) value increased exponentially with decreasing [Ca(2+)], with an e-fold change observed per 0.69 mM decrease in [Ca(2+)]. Loading neurons with BAPTA abolished Ca(2+)-dependent inhibition, suggesting that Ca(2+) affects NMDARs from the cytosol. Since there is one known Ca(2+)-dependent process in gating of NMDARs, we conclude that ATL most likely promotes Ca(2+)-dependent desensitization. We also found ATL to be a trapping open-channel blocker of NMDARs with an IC(50) of 220 µM at 0 mV. An e-fold change in ATL IC(50) was observed to occur with a voltage shift of 50 mV in 0.25 mM [Ca(2+)]. Thus, we disclose here a robust dependence of ATL potency on extracellular [Ca(2+)], and demonstrate that ATL bound in the NMDAR pore can be trapped by closure of the channel.