The Na+ channel alpha-subunit contains an IFM motif that is critical for the fast inactivation process. In this study, we sought to determine whether an IFM-containing peptide, acetyl-KIFMK-amide, blocks open cardiac Na+ channels via the inner cavity. Intracellular acetyl-KIFMK-amide at 2 mM elicited a rapid time-dependent block (tau = 0.24 ms) of inactivation-deficient human heart Na+ channels (hNav1.5-L409C/A410W) at +50 rnV. In addition, a peptide-induced tail current appeared conspicuously upon repolarization, suggesting that the activation gate cannot close until acetyl-KIFMK-amide is cleared from the open pore. Repetitive pulses (+50 mV for 20 ins at 1 Hz) produced a substantial use-dependent block of both peak and tail currents by similar to 65%. A F1760K mutation (hNav1.5-L409C/A410W/F1760K) abolished the use-dependent block by acetyl-KIFMK-amide and hindered the time-dependent block. Competition experiments showed that acetyl-KIFMK-amide antagonized bupivacaine binding. These results are consistent with a model that two acetyl-KIFMK-amide receptors exist in proximity within the Na+ channel inner cavity. (c) 2005 Elsevier Inc. All rights reserved.