Site-Directed Spin Labeling Reveals Pentameric Ligand-Gated Ion Channel Gating Motions

Abstract

Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, binding of agonist to the extracellular domain triggers a structural rearrangement that leads to the opening of an ion-conducting pore in the transmembrane domain and, in the continued presence of neurotransmitter, the channels desensitize (close). The flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7, and 9) to the transmembrane channel domain (M2–M3 loop) are essential for coupling ligand binding to channel gating. Comparing the crystal structures of two bacterial pLGIC homologues, ELIC and the proton-activated GLIC, suggests channel gating is associated with rearrangements in these loops, but whether these motions accurately predict the motions in functional lipid-embedded pLGICs is unknown. Here, using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and functional GLIC channels reconstituted into liposomes, we examined if, and how far, the loops at the ECD/TMD gating interface move during proton-dependent gating transitions from the resting to desensitized state. Loop 9 moves ∼9 Å inward toward the channel lumen in response to proton-induced desensitization. Loop 9 motions were not observed when GLIC was in detergent micelles, suggesting detergent solubilization traps the protein in a nonactivatable state and lipids are required for functional gating transitions. Proton-induced desensitization immobilizes loop 2 with little change in position. Proton-induced motion of the M2–M3 loop was not observed, suggesting its conformation is nearly identical in closed and desensitized states. Our experimentally derived distance measurements of spin-labeled GLIC suggest ELIC is not a good model for the functional resting state of GLIC, and that the crystal structure of GLIC does not correspond to a desensitized state. These findings advance our understanding of the molecular mechanisms underlying pLGIC gating.

The M4 Transmembrane α-Helix Contributes Differently to Both the Maturation and Function of Two Prokaryotic Pentameric Ligand-gated Ion Channels

“…Regardless, the data suggest that there may be distinct features in the mechanisms of activation in GLIC and ELIC, a finding supported by the observation that ELIC exhibits atypical gating and cation-conduction properties relative to other pLGICs (59). Double electron-electron resonance experiments also suggest that the x-ray structures of ELIC are not appropriate models for the resting state (60). Taken together, these findings caution against the utility of comparing different conformations of GLIC and ELIC to probe the mechanisms underlying agonist-induced gating.…”

Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC

“…The DEER experiment has shown to be a valuable technique to measure inter-spin distances within membrane proteins (Dellisanti et al, 2013; Jeschke, 2012; Jeschke et al, 2004). In particular, for a pentameric assembly containing single-labeled subunits, a bimodal distribution (Figure 5a and 5b) consisting of an adjacent and non-adjacent distance is expected (Dalmas et al, 2012).…”

“…The debate about what causes non-conductivity in the pore of desensitized pLGICs continues, despite the fact that several structural models have been proposed to describe pLGIC desensitization (Dellisanti et al, 2013; Keramidas and Lynch, 2013; Velisetty and Chakrapani, 2012; Wang and Lynch, 2011; Zhang et al, 2011). Further structural investigation is necessary to evaluate these models and to determine whether conformational rearrangement is uniform across all pLGICs or each pLGIC has its own unique structural change under desensitization.…”

“…Crystal structures of ELIC in the resting state have been determined to ~3 Å resolutions (Hilf and Dutzler, 2008; Pan et al, 2012b), but co-crystallization of ELIC with its agonists has not yielded high-resolution structures that can unambiguously differentiate the resting and desensitized ELIC (Spurny et al, 2012; Zimmermann and Dutzler, 2011). 19 F NMR (Kitevski-LeBlanc and Prosser, 2012; Liu et al, 2012) is a valuable addition to crystallography and other approaches used for characterizing the structures and dynamics of pLGICs (Cymes and Grosman, 2008; Dellisanti et al, 2013; Purohit et al, 2013; Sauguet et al, 2014; Unwin and Fujiyoshi, 2012; Velisetty and Chakrapani, 2012; Velisetty et al, 2012, 2014). The major findings from the present study include: (1) a new NMR model for the desensitized ELIC with an expansion at the upper half of the pore and a contraction at the lower half of the pore, which is further supported by ESR data; (2) co-existence of multiple conformations of ELIC in both the resting and desensitized states; and (3) non-uniform and asymmetric conformational rearrangements accompanying the channel’s transition from the resting to desensitized state.…”

The Synergetic Effects of Combining Structural Biology and EPR Spectroscopy on Membrane Proteins

“…Besides nowadays various structures a plethora of EPR studies has been performed with regard to conformational changes in the selectivity filter [65,66], the cytoplasmic helical bundle [67,68] and the communication between both features [69]. Further excellent examples for the combined use of structural biology and EPR spectroscopy are present for all classes of ion channels, e.g., for the intracellular Mg 2+ -gated Mg 2+ channel CorA [70][71][72], the nucleotide-and Na + -gated K + channel KtrAB [73], the mechanosensitive channel MscL [74,75], the cyclic AMP-gated HCN channel [45,76], the voltage-gated Na + channel NavMs [77] and the pentameric receptor channels GLIC [78]. In all cases EPR measurements have been applied to validate existing protein structures, determine the structure of missing regions or distorted elements in structures, or add valuable information of the dynamics of the studied systems to provide a model of activation and regulation.…”