Amitriptyline is a tricyclic antidepressant used to treat major depression and various neuropathic pain syndromes. This drug also causes cardiac toxicity in patients with overdose. We characterized the tonic and use-dependent amitriptyline block of human cardiac (hH1) Na(+) channels expressed in human embryonic kidney cells under voltage-clamp conditions. Our results show that, near the therapeutic plasma concentration of 1 microM, amitriptyline is an effective use-dependent blocker of hH1 Na(+) channels during repetitive pulses (approximately 55% block at 5 Hz). The tonic block for resting and for inactivated hH1 channels by amitriptyline (0.1-100 microM) yielded IC(50) values (50% inhibitory concentration) of 24.8 +/- 2.0 (n = 9) and 0.58 +/- 0.03 microM (n = 7), respectively. Substitution of phenylalanine with lysine at the hH1-F1760 position, a putative binding site for local anesthetics, eliminates the use-dependent block by amitriptyline at 1 microM. The time constants of recovery from the inactivated-state amitriptyline block in hH1 wild-type and hH1-F1760K mutant channels are 8.0 +/- 0. 5 (n = 6) and 0.45 +/- 0.07 s (n = 6), respectively. A substitution at either hH1-F1760K or hH1-Y1767K significantly increases the IC(50) values for resting and inactivated states of amitriptyline, but the increase is much more pronounced with the hH1-F1760K mutation. Because these two residues were proposed to form a part of the local anesthetic binding site, we conclude that amitriptyline and local anesthetics interact with a common binding site. Furthermore, at therapeutic concentrations, the ability of amitriptyline to act as a potent use-dependent blocker of Na(+) channels may, in part, explain its analgesic actions.