Small Molecule\nNS11021 Promotes BK Channel Activation\nby Increasing Inner Pore Hydration

The\nCa<sup>2+</sup> and voltage-gated big potassium (BK)\nchannels\nare implicated in various diseases, including heart disease, asthma,\nepilepsy, and cancer, but remain an elusive drug target. A class of\nnegatively charged activators (NCAs) have been demonstrated to promote\nthe activation of several potassium channels including BK channels\nby binding to the hydrophobic inner pore, yet the underlying molecular\nmechanism of action remains poorly understood. In this work, we analyze\nthe binding mode and potential activation mechanism of a specific\nNCA named NS11021 using atomistic simulations. The results show that\nNS11021 binding to the pore in deactivated BK channels is nonspecific\nand dynamic. The binding free energy of −8.3 ± 0.7 kcal/mol\n(<i>K</i><sub><i>D</i></sub> = 0.3–3.1\nμM) calculated using umbrella sampling agrees quantitatively\nwith the experimental EC50 range of 0.4–2.1 μM. The bound\nNS11021 remains dynamic and is distal from the filter to significantly\nimpact its conformation. Instead, NS11021 binding significantly enhances\nthe pore hydration due to the charged tetrazol-phenyl group, thereby\npromoting the opening of the hydrophobic gate. We further show that\nthe free energy barrier to K<sup>+</sup> permeation is reduced by\n∼3 kcal/mol regardless of the binding pose, which could explain\nthe ∼62-fold increase in the intrinsic opening of BK channels\nmeasured experimentally. Taken together, these results support the\nidea that the molecular mechanism of NS11021 derives from increasing\nthe hydration level of the conformationally closed pore, which does\nnot depend on specific binding and likely explains the ability of\nNCAs to activate multiple K<sup>+</sup> channels.