Molecular Basis of Dual-Site Engagement and Selectivity of CcoTx3-synth for hNaV1.5.

Voltage-gated sodium channels (NaVs) are critical membrane proteins in excitable cells, with NaV1.5 playing a pivotal role in cardiac electrophysiology. Mutations of human NaV1.5 (hNaV1.5) are linked to severe cardiac channelopathies, including atrial fibrillation and Brugada syndrome, making it a prime therapeutic target. Natural peptide toxins from venomous animals, such as tarantulas, offer valuable molecular tools for exploring NaV function. Among these, CcoTx3, a toxin from the straight-horned baboon tarantula, selectively inhibits hNaV1.5/β1 (IC50 = 447 nM), while other NaV subtypes are unaffected (hNaV1.1/β1-1.4/β1) or only partially blocked at higher concentrations (hNaV1.8/β1). Despite its pharmacological potential, the structural basis of CcoTx3 interaction with hNaV1.5 remains unresolved, particularly for the resting-state voltage-sensing domains II (VSDII) and IV (VSDIV), for which no experimental structure exists. This study combined molecular modeling and site-directed mutagenesis to elucidate the binding mechanism of the synthetic CcoTx3-hNaV1.5. Models of CcoTx3 bound to VSDII and VSDIV were generated, and molecular interactions were analyzed. Key binding residues were validated through site-directed mutagenesis at neurotoxin sites 3 and 4, followed by two-electrode voltage clamp assays. S743, E746, E747, R800, and S802 were identified as critical hotspots for inhibition of channel activation in VSDII, while D1610 and K1614 were key for fast inactivation inhibition. Synthetic CcoTx3 toxin (CcoTx3-synth) also interacted with zwitterionic and negatively charged liposomes, supporting a reduction-of-dimensionality mechanism. These findings provide the first structural and functional insights into the selective multisite engagement of CcoTx3-synth with hNaV1.5 and may guide the design of peptide therapeutics targeting cardiac channelopathies.