Using simulation to probe physiology and pharmacology of ion channels

Thesis

In ion channels, fenestrations are lateral openings that connect the membrane to the protein’s ion-conducting pore. They are well known to act as access pathways for lipophilic drugs in voltage-gated sodium channels and have been implicated in inheritable diseases. Despite their clear functional importance in many ion channel families, no such fenestrations have previously been reported in the superfamily of pentameric ligand-gated ion channels (pLGICs). This channel family has been the subject of in-depth study for decades, but many questions on the precise details of channel gating and pharmacology still remain. The glycine receptor (GlyR) is an inhibitory pLGIC that is responsible for regulation of motor control in the spinal cord. Its activation and drug interactions are the focus of this thesis.

Here, I developed an improved umbrella sampling workflow aimed at improving convergence speeds of potentials of mean force (PMF). I benchmarked this workflow on test systems of drug molecules permeating through lipid bilayers of different lipid compositions. I found that my improved workflow outperforms common practices in umbrella sampling, and also quantified errors arising from boundary conditions commonly applied to the calculation of PMFs.

The main finding of this DPhil is the identification of a state-dependent, drugpermeable fenestration in GlyR. I analysed 55 published GlyR structures in different functional states for potential fenestrations and consistently detected a statedependent fenestration near an anaesthetics binding site at each subunit interface. Observed to be almost exclusively occluded in the closed state, the fenestration widens considerably in open state structures, exhibiting a similar ‘bottleneck’ radius across structures. I then employed my improved umbrella sampling workflow to quantify the translocation energetics of fipronil. Comparison of the resulting potential of mean force (PMF) profiles shows that fenestration-based entry of fipronil is energetically favourable to membrane permeation. My results thus provide the first characterisation of a drug-permeable fenestration in GlyR.

Additionally, through investigations of precisely how binding of the positive allosteric modulator ivermectin destabilises the orthosteric inhibitor strychnine, allosteric networks in GlyR that tie conserved ligand binding loops to the transmembrane domain were described. Finally, intermediate activation states of the Gloeobacter ligand-gated ion channel (GLIC) were probed in computational electrophysiology. We demonstrate that intermediate, asymmetric activation states of the channel already display partial conductivity.

Authors: Feddersen, B

• DOI: 10.5287/ora-eor0kpxdo
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: molecular dynamics; rule of 5; ion channel; pentameric ligand-gated ion channel; MD; CAVER; glycine receptor; drug access; membrane permeation; potential of mean force; free energy; PMF; gloeobacter; logP; fenestration; GLIC
• Subjects: Biochemistry; Ligand binding (Biochemistry); Biophysics; Structural bioinformatics