State-dependent block of voltage-gated Na+ channels by amitriptyline via the local anesthetic receptor and its implication for neuropathic pain

Amitriptyline is a tricyclic anti depressant, which also alleviates various pain syndromes at its therapeutic plasma concentration (0.36-0.90 muM). Accumulated evidence suggests that such efficacy may be due to block of voltage-gated Na+ channels. The Na+ channel alpha-subunit protein consists of four homologous domains (D1-D4), each with six transmembrane segments (S1-S6). The aims of this study were to locate the amitriptyline receptor in the Na+ channel a-subunit and to compare the amitriptyline affinity in open, inactivated, and resting states of the Na+ channel. Wild-type and mutant rat skeletal muscle alpha-subunit Na+ channels were expressed in human embryonic kidney cells and assayed under whole-cell voltage clamp conditions. Our results indicate that the amitriptyline receptor overlaps with the local anesthetic receptor to a great extent in Na+ channels. Residues N434 (at D1-S6), L1280 (D3-S6), and F1579 (D4-S6) may jointly form parts of the amitriptyline/local anesthetic receptor, with residue L1280 being most critical for amitriptyline binding. Open-channel block by amitriptyline was assessed in inactivation-deficient Na+ channels and compared with the resting- and inactivated-channel block in wild-type channels. The open-channel block by amitriptyline has the highest affinity, with a 50% inhibitory concentration (IC50) of 0.26 muM. The inactivated-channel block by amitriptyline had a weaker affinity (0.51 muM), whereas the resting-channel displayed the weakest affinity (33 muM). We hypothesize that selective block of both persistent late openings and the inactivated state of neuronal Na+ channel isoforms by amitriptyline also occurs at its therapeutic concentration and likely contributes to its efficacy in pain syndromes. (C) 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.