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Microsecond-timescale simulations suggest 5-HT–mediated preactivation of the 5-HT3A serotonin receptor

Nicholas B. Guros,Arvind K. Balijepalli,Jeffery B. Klauda

Published 2019 in Proceedings of the National Academy of Sciences of the United States of America

ABSTRACT

Significance The pentameric ligand-gated ion channel 5-HT3A exhibits a transient preactive state that provides valuable insight into its binding and activation mechanisms. In this preactive state, the allosteric regulation of the channel structure, and thereby function, upon ligand binding occur at timescales that are too fast to be measured experimentally. This study demonstrates the power of microsecond-timescale MD simulations in detecting these transitional preactive states upon ligand binding and describing their effects on channel function at the molecular scale. Such a mechanistic understanding of the channel function is a critical element in the design of therapeutics for the regulation of 5-HT3A, which are needed to reverse the effects of numerous pathological conditions. Aided by efforts to improve their speed and efficiency, molecular dynamics (MD) simulations provide an increasingly powerful tool to study the structure–function relationship of pentameric ligand-gated ion channels (pLGICs). However, accurate reporting of the channel state and observation of allosteric regulation by agonist binding with MD remains difficult due to the timescales necessary to equilibrate pLGICs from their artificial and crystalized conformation to a more native, membrane-bound conformation in silico. Here, we perform multiple all-atom MD simulations of the homomeric 5-hydroxytryptamine 3A (5-HT3A) serotonin receptor for 15 to 20 μs to demonstrate that such timescales are critical to observe the equilibration of a pLGIC from its crystalized conformation to a membrane-bound conformation. These timescales, which are an order of magnitude longer than any previous simulation of 5-HT3A, allow us to observe the dynamic binding and unbinding of 5-hydroxytryptamine (5-HT) (i.e., serotonin) to the binding pocket located on the extracellular domain (ECD) and allosteric regulation of the transmembrane domain (TMD) from synergistic 5-HT binding. While these timescales are not long enough to observe complete activation of 5-HT3A, the allosteric regulation of ion gating elements by 5-HT binding is indicative of a preactive state, which provides insight into molecular mechanisms that regulate channel activation from a resting state. This mechanistic insight, enabled by microsecond-timescale MD simulations, will allow a careful examination of the regulation of pLGICs at a molecular level, expanding our understanding of their function and elucidating key structural motifs that can be targeted for therapeutic regulation.

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