Abstract Body (Do not enter title and authors here): INTRODUCTION: Bupivacaine (BPV) is a lipophilic local anesthetic (LA) that blocks voltage-gated sodium channels (Na_v) to prevent pain and provide analgesia during nerve blocks and epidural anesthesia. Though generally safe, BPV has the narrowest therapeutic window of all LAs and has been associated with fatal adverse events due to cardiotoxicity. Previous investigators have shown these events are due to BPV’s slow off-rate relative to other LAs, though the reasons for its slow off-rate are unknown, as is BPV’s binding site in cardiac Na_v (Na_v1.5). Here we have investigated the structural basis of BPV’s cardiotoxicity by using cryogenic electron microscopy (cryo-EM) and electrophysiology to determine BPV’s binding site in Na_v1.5. These data allow comparison with other local anesthetics and provide the first step toward eliminating BPV’s cardiotoxicity.

HYPOTHESIS: BPV’s cardiotoxicity relative to other LAs is a result of unique binding pose in Na_v1.5.

APPROACH: Cryo-EM data were collected from vitrified protein samples, then analyzed using CryoSparc. 3D reconstructions were fit with a model of Na_v1.5 in complex with BPV, then refined and validated using Phenix and Coot. Electrophysiology data were recorded by transfecting TsA201 cells with Na_v1.5-eGFP and measuring drug effect using voltage-clamp. Tonic block was measured by a long pulse to open the channel at 0.2Hz, whereas use-dependent block was recorded using a short pulse protocol at 10Hz. Statistics were performed using GraphPad Prism 8.0.2.

RESULTS: BPV binds to Na_v1.5 by p-stacking with the residue F1760 and making hydrophobic contacts with its unique alkyl chain. Recordings of wild-type Na_v1.5 and Na_v1.5-F1760A under voltage-clamp confirm the role of F1760 in in block of Na_v1.5 by BPV as the F1760A mutation reduces tonic block by 81.63% (54.66% 2.42% in WT vs 10.04% 9.30% in F1760A) and use-dependent block by 95.84% (79.29% 5.70% in WT vs 3.30% 7.64% in F1760A) in the presence of 100 µM BPV. Comparison of BPV’s pose to that of other drugs highlights the role of its unique alkyl chain in stabilizing binding and delaying recovery, which are key factors in the development of cardiotoxicity.

CONCLUSIONS: BPV’s slow off-rate from Na_v1.5 derives from its unique alkyl chain, which is not necessary for lipophilicity or drug action. These data highlight the potential for modified, lipophilic LAs which retain high potency in nerve without causing cardiotoxicity.