DIV/S3-S4 loop length determines Nav1.8 insensitivity to the scorpion α-like toxin BmK I

Nav1.8, a voltage-gated sodium channel predominantly expressed in dorsal root ganglion neurons, is a key mediator of pain signaling and an established therapeutic target. However, its direct interaction with BmK I, an α-like scorpion toxin considered to be the primary factor in scorpion sting pain, has remained uncertain. By whole-cell patch-clamp recordings, we evaluated the sensitivity of rat Nav1.8 (rNav1.8) to BmK I and generated a panel of rNav1.5-based mutants to investigate the functional role of two extra residues (leucine-glutamate, LE) in the DIV/S3-S4 extracellular loop. At 300 nM concentration, BmK I did not significantly slow the inactivation of rNav1.8 (with no significant change in I₅ms/I_peak, P > 0.05). Insertion of LE, or its substitution with leucine-glutamine (LQ), glutamate-histidine (EH), aspartate (D), alanine-alanine (AA), or alanine (A), at the equivalent position of rNav1.5 consistently attenuated BmK I efficacy, yet none of the mutants became completely insensitive. Notably, insertion of alanine produced a similar effect, implicating steric hindrance, rather than side-chain chemistry, as the primary determinant. Conversely, deletion of LE from rNav1.8 fully restored BmK I sensitivity. In marked contrast, the classical α-toxin AaH II still markedly delayed inactivation of both rNav1.5-LE and rNav1.5-AA, revealing that BmK I and AaH II rely on distinct conformational features within receptor site-3. Together, these findings indicate that the length of the DIV/S3-S4 loop, rather than the chemical identity of the inserted residues, is the primary factor correlating with rNav1.8 resistance to BmK I. This correlation is consistent with a steric hindrance mechanism, although direct structural evidence is not provided by the current functional data. Nonetheless, our results offer structural clues for the development of subtype-selective sodium channel modulators.