CONICET | Buscador de Institutos y Recursos Humanos

Bupivacaine (B) is a local anesthetic that belongs to the amino amide group (B/BH+ equilibrium, pKa=8.1). Previously, we demonstrated [1] that bupivacaine inhibits the potassium channel BKCa, by a very-fast type block and voltage-dependent mechanism (depolarization increases the blocking ratio). This in hibition occurs from the intracellular side, by interacting with the channel pore. In this study we investigated the effect of pH (pH= 7.4 and pH= 7.0) on bupivacaine blocking action on BKCa channel expressed in HEK293T cells. In addition, we studied the drug-protein interaction mechanism (B and BH+), using the computational docking technique.Using the patch clamp technique in voltage clamp configuration, G/Gmax curves were built in absence and in the presence of 300μM bupivacaine at both pH values. The analysis of the steady-state ionic currents showed that, at pH= 7.0 (BH+/B=12.6), the addition of bupivacaine generated a 50% decrease in Gmax. At pH: 7.4 (BH+/B= 5) the decrease in Gmax was 25%. Moreover, the bupivacaine had no effect on the instantaneous current, measured on tail currents. We also performed docking simulations of BH+ and B in the BKCa channel homology model obtained before by our group. The grid where the docking was performed contained the pore structure of the BKCa channel. The lowest energy conformations of the ligands were docked below the selectivity filter interacting with the residues L377, F380, and V384. BH+ also interacts with T352 of the selectivity filter. These results confirm that the blocking effect is greater when the ratio of BH+/B was higher and that it is mediated by a voltage-dependent blockade of the channel unitary conductance without affecting the channel opening probability.1. Martin, P. et al. Channels (Austin). 2012. p174-80