Interaction of local anesthetics with a peptide encompassing the IV/S4-S5 linker of the Na+ channel

The peptide pIV/S4-S5 encompasses the cytoplasmic linker between helices S4-S5 in domain IV of the voltage-gated Na⁺ channel, residues 1644-1664.²2Residues numbering correspond to the primary sequence of the voltage-gated human brain Na⁺ channel [1]. The interaction of two local anesthetics (LA), lidocaine and benzocaine, with pIV/S4-S5 has been studied by DOSY, heteronuclear NMR ¹H-¹⁵N-HSQC spectroscopy and computational methods. DOSY indicates that benzocaine, a neutral ester, exhibits stronger interaction with pIV/S4-S5 than lidocaine, a charged amine-amide. Weighted average chemical shifts, Δδ(¹H-¹⁵N), show that benzocaine affects residues L¹⁶⁵³, M¹⁶⁵⁵ and S¹⁶⁵⁶ while lidocaine slightly perturbs residues I¹⁶⁴⁶, L¹⁶⁴⁹ and A¹⁶⁵⁹, L¹⁶⁶⁰, near the N- and C-terminus, respectively. Computational methods confirmed the stability of the benzocaine binding and the existence of two binding sites for lidocaine. Even considering that the approach of studying the peptide in the presence of a co-solvent (TFE/H₂O, 30%/70% v/v) has an inherently limited implication, our data strongly support the existence of multiple LA binding sites in the IV/S4-S5 linker, as suggested in the literature. In addition, we consider that LA can bind to the S4-S5 linker with diverse binding modes and strength since this linker is part of the receptor for the "inactivation gate particle". Conditions for devising new functional studies, aiming to better understand Na⁺ channel functionality as well as the various facets of LA pharmacological activity are proposed in this work.