Small Molecule NS11021 Promotes BK Channel Activation by Increasing Inner Pore Hydration

The Ca2+ and voltage-gated big potassium (BK) channels are implicated in various diseases, including heart disease, asthma, epilepsy, and cancer, but remain an elusive drug target. A class of negatively charged activators (NCAs) have been demonstrated to promote the activation of several potassium channels including BK channels by binding to the hydrophobic inner pore, yet the underlying molecular mechanism of action remains poorly understood. In this work, we analyze the binding mode and potential activation mechanism of a specific NCA named NS11021 using atomistic simulations. The results show that NS11021 binding to the pore in deactivated BK channels is nonspecific and dynamic. The binding free energy of −8.3 ± 0.7 kcal/mol (K D = 0.3–3.1 μM) calculated using umbrella sampling agrees quantitatively with the experimental EC50 range of 0.4–2.1 μM. The bound NS11021 remains dynamic and is distal from the filter to significantly impact its conformation. Instead, NS11021 binding significantly enhances the pore hydration due to the charged tetrazol-phenyl group, thereby promoting the opening of the hydrophobic gate. We further show that the free energy barrier to K+ permeation is reduced by ∼3 kcal/mol regardless of the binding pose, which could explain the ∼62-fold increase in the intrinsic opening of BK channels measured experimentally. Taken together, these results support the idea that the molecular mechanism of NS11021 derives from increasing the hydration level of the conformationally closed pore, which does not depend on specific binding and likely explains the ability of NCAs to activate multiple K+ channels.