Previously, domain Ⅳ S3/S4 linker was demonstrated to be an important region determing the sensitivity of sodium channels to BmK Ⅰ, a sodium channel-specific site-3 modulator.But the localization of molecular determinants in sensitivity of sodium channels to BmK Ⅰ remained unclear.In this study, through channel alignment, DIV/S3-S4 linker in BmK Ⅰ-insensitive rNav1.8 was inserted by two amino acids (LE) compared with rNav1.5.By constructing channel mutants mimicing the rNav1.8 DIV S3/S4 region in rNav1.5 backbone, the sensitivity of rNav.15 mutants to BmK Ⅰ was remarkably reduced.Furthermore, two peptides were synthesized according to the DIV/S3-S4 linker in rNav1.5 (peptide P1) and rNav1.8 (peptide P2).The binding affinities of peptides to BmK Ⅰ was investigated by surface Plasmon resonance (SPR).The results showed that both P1 and P2 could bind with BmK Ⅰ.But there were significantly differences in binding capability between these two peptides.The binding response (RU) of the peptide P2 was reduced to about 1/3 of the peptide P1.The association kinetics of P2 was also accelerated.It was presumed that the insensitivity of sodium channels to BmK I could be attributed to the alteration of microstructure of the receptor site by LE insertion.Thus, it may provide cludes for the diversity of spatial structure of receptor sites mapping for BmK Ⅰ-sodium channel interaction.