The S6 segment of domain IV (DIV-S6) of the voltage-gated Na channel is considered to be a key player in gating and local anesthetic drug block. Thus, some mutations in DIV-S6 substantially alter the channel's inactivation properties. In order to get a comprehensive picture of the kinetic role of DIV-S6 in fast inactivation we performed a cysteine scanning analysis of sites 1575-1591 in the DIV-S6 of the rNav1.4 channel. In addition, we produced the same cysteine replacements in the background of the mutation K1237E. K1237 is located in the P-loop of domain III and mutations at this site have dramatic effects both on permeation and gating properties. Hence, K1237E most likely causes a complex conformational change of the channel. We sought to explore whether K1237E changes the pattern of gating perturbations by the serial cysteine replacements in DIV-S6. The constructs were expressed in Xenopus laevis oocytes and studied by means of two electrode voltage-clamp. The half-point of availability following a 50 ms conditioning prepulse (V05) was -44 ± 1 mV and -51 ± 1 mV in wild-type and K1237E, respectively (P < 0.001) . Most serial amino acid replacements in DIV-S6 produced shifts in V05, both in wild-type and in K1237E background, ranging from +17 ± 1 mV to -9 ± 2 mV. A plot of the shifts in V05 by single DIV-S6 mutants relative to wild-type versus the shifts in V05 by double mutants relative to K1237E showed a significant positive correlation (R= 0.72, P=0.002). This indicates that the general pattern of gating perturbations in DIV-S6 is not affected by K1237E, suggesting a high conformational stability of the DIV-S6 segment during the fast inactivated state. Support: FWF P21006-B11