Abstract
Ligand-gated ion channels conduct currents in response to chemical stimuli, mediating electrochemical signaling in neurons and other excitable cells. For many channels, the details of gating remain unclear, partly due to limited structural data and simulation timescales. Here, we used enhanced sampling to simulate the pH-gated channel GLIC, and construct Markov state models (MSMs) of gating. Consistent with new functional recordings, we report in oocytes, our analysis revealed differential effects of protonation and mutation on free-energy wells. Clustering of closed- versus open-like states enabled estimation of open probabilities and transition rates, while higher-order clustering affirmed conformational trends in gating. Furthermore, our models uncovered state- and protonation-dependent symmetrization. This demonstrates the applicability of MSMs to map energetic and conformational transitions between ion-channel functional states, and how they reproduce shifts upon activation or mutation, with implications for modeling neuronal function and developing state-selective drugs.
Highlights
The family of pentameric ligand-gated ion channels, known as Cys-loop receptors, controls electrochemical signal transduction in numerous tissues and cell types, from bacteria to humans
In addition to capturing features of the gating mechanism already proposed for pentameric ligand-gated ion channels (pLGICs), our Markov state models (MSMs) allowed for further exploration of features that correlate with gating
We focused on the effect of conformational symmetry between subunits and found that the open state displayed higher levels of symmetry, which was enhanced after protonation (Figure 7C)
Summary
The family of pentameric ligand-gated ion channels (pLGICs), known as Cys-loop receptors, controls electrochemical signal transduction in numerous tissues and cell types, from bacteria to humans. Application of the string method with swarms of trajectories recently enabled the identification of local rearrangements in channel closure, including contraction of the upper pore, loosening of β-strand contacts in the lower ECD, and general expansion of the upper ECD (Lev et al, 2017) This provides precious information of structural rearrangements, but the choice of collective variables in combination with the timescales of individual simulations may influence what motions are sampled. We have used such enhanced seeding approaches combined with MSMs to sample the GLIC opening-closing transition Both the wild-type and two different variants with mutations along the pore-lining M2 helices - where we showed one to yield gain-of-function similar to human homologs (Filatov and White, 1995; Kosolapov et al, 2000) and the other loss of function (Fourati et al, 2018) - were simulated in resting or activating conditions (neutral or low pH). We present new evidence for the role of symmetry in gating
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