The quaternary ammonium (QA) lidocaine derivative QX‐314 (2‐(triethylamino)‐N‐(2,6‐dimethylphenyl)‐acetamide) induces internal pore blockade of single cardiac Na⁺ channels enzymatically modified (papain) to eliminate fast inactivation. The mechanism involves dual, interacting blocking modes (rapid and discrete) with binding domains deep in the pore from the cytoplasmic mouth, and where the rapid blocked configuration serves as a transition‐intermediate for the development of discrete block. The primary goals of this study were to test for this mechanism in a recombinant Na⁺ channel genetically engineered to selectively lack fast inactivation, and if present, to explore the underlying structural features.

Fast inactivation was removed in rat skeletal muscle μ1 Na⁺ channels (RSkM1) with an IFM‐QQQ mutation in the cytoplasmic III‐IV interdomain (QQQ). QQQ was expressed in Xenopus oocytes and single‐channel activity was studied in cell‐free, inside‐out membrane patches. Application of QX‐314 (QX, 0‐4 mM) to the cytoplasmic membrane surface caused two distinct modalities of single‐channel blockade: reduction of unitary current and interruptions of current lasting tens of milliseconds. These are consistent with rapid and discrete pore block, respectively. The voltage and concentration dependence of block indicates that the modes interact and have binding sites that share a deep location in the pore, at ≈65 % of the membrane electric field in from the cytoplasmic mouth.

Mutation of phenylalanine (F1579) in domain IV‐S6, critical in local anaesthetic block, to alanine in QQQ (QQQ‐F1579A) disabled discrete block but notably failed to alter rapid block, single‐channel gating and slope conductance.

Amplitude distribution analysis was applied to long bursts (> 50 ms) of QQQ‐F1579A activity to investigate the kinetics of rapid block. Computed rapid blocking and unblocking rate constants are 42 000 ± 18 000 M⁻¹ ms⁻¹ and 82 ± 22 ms⁻¹, respectively (n= 3, ‐20 mV).

The results support a general transition‐intermediate mechanism that governs internal QX and local anaesthetic pore block of voltage‐gated Na⁺ channels and provide insight into underlying structural features.