Agonist Discovery And Pathogenic Mutation Molecular Mechanism Study Of KCNQ Channels

Voltage-gated potassium channels KCNQ(KCNQ1–KCNQ5)play important roles in regulating neuronal excitability.KCNQ gene mutations can lead to a series of neurological disorders such as epilepsy,autism and intellectual deficiency.However,currently there are no safe and effective drug molecules available which could target KCNQ channels for therapy.And there are also no personalized treatments for specific KCNQ gene pathogenic mutations.Moreover,the molecular mechanisms behind most KCNQ gene mutations which can cause channel dysfunction are still unknown.How to find new modulators with novel action mechanism targeting KCNQ channels,and what the molecular mechanisms are behind KCNQ pathogenic mutations,are two urgent scientific problems to be solved in KCNQ channel pharmacology.Structure-based drug design and molecular dynamics simulation are two important means for drug discovery and study of the relationship between protein structure and function.They can identify new drug-like molecules for specific targets and explore the molecular mechanism of amino acid mutations affecting protein function at the atomic level.In this thesis,by using the above methods combined with electrophysiological experiments we have discovered a novel agonist of the KCNQ channels and explored the molecular mechanism of a KCNQ mutation which can lead to channel dysfunction.Under the known pharmacological activation mechanisms,agonists open channels by directly or allosterically targeting potassium channel gates.In the first and second part of the thesis,we proposed a new pharmacological regulation strategy to target the central cavity of the KCNQ2 channel.And using the structure-based drug design method,we discovered a novel agonist CLE030 which can directly regulate the permeanting ions.Unlike other reported KCNQ2 agonists,CLE030 enhances the voltage-activated KCNQ2 current at all tested voltages without changing the voltage sensitivity of the channel.Site-directed mutagenesis,molecular docking,derivatives design and electrophysiological experiments collectively confirm that CLE030 binds in the central cavity.Molecular dynamics simulations and free energy calculations reveal that CLE030 adopts an atypical gate-independent pharmacological activation mechanism in which small molecules increase channel currents by helping potassium ions pass through the central cavity.In addition,this atypical pharmacological activation mechanism is also feasible to the filter-gated potassium channel TREK-1.KCNQ2 and KCNQ3 are assembled into heteromeric channels,which mediate M currents and jointly determine neuronal subthreshold excitability.Thousands of KCNQ3 gene mutations have been reported to be related to human diseases.But the molecular mechanism of these mutations is rarely studied.In the third part of the thesis,we used molecular dynamics simulation to explore the molecular mechanism of channel dysfunction caused by R230 C mutation in KCNQ3 channel,which is associated with severe neurodevelopmental disorders.We found that R230 C mutation did not affect the activated conformation of voltage-sensing domain(VSD).However,the R230 C mutation with a reduced side chain size makes the VSD more likely to approach the pore domain(PD)and maintain a close interaction with the PD of the channel.And R230 C mutation could result a water-permeating pathway in the VSD and increase the hydration level around the gating charge residues.These may be the reason why KCNQ3-R230 C mutation channels are more difficult to deactivate and thus remain constitutively open.This study not only helps to reveal the molecular mechanism behind KCNQ3 mutation,but also provides clues for more effectively targeting this mutation.In conclusion,our study not only discovered a novel KCNQ agonist with new skeleton structure,new binding site and new action mechanism,but also revealed the molecular mechanism of channel dysfunction caused by KCNQ3 R230 C mutation,which both layed the foundation for the development of more small molecules targeting KCNQ channels.