KPQ, a three amino acid [lysine (K), proline (P), glutamine (Q)] deletion mutation of the human cardiac Na channel (hH1), which is one cause of long QT syndrome (LQT3), has impaired inactivation resulting in a late sodium current. To better understand inactivation in KPQ, we applied a site-3 toxin anthopleurin A, which has been shown to inhibit inactivation from the open state with little or no effect on inactivation from the closed state(s) in wild-type hH1. In contrast to the effect of site-3 toxins on wild-type hH1, inactivation from closed state(s) in toxin-modified KPQ demonstrated a large negative shift in the Na channel availability curve of nearly 14 mV. Recovery from inactivation showed that toxin-modified KPQ channels recovered slightly faster than those in control, whereas development of inactivation at potentials negative to 80 mV showed that inactivation developed much more rapidly in toxin-modified KPQ channels compared with control. An explanation for our results is that closed-state inactivation in toxin-modified KPQ is enhanced by the mutated inactivation lid being positioned "closer" to its receptor resulting in an increased rate of association between the inactivation lid and its receptor.