Signal Transduction at the Domain Interface of Prokaryotic Pentameric Ligand-Gated Ion Channels

Abstract

Pentameric ligand-gated ion channels are activated by the binding of agonists to a site distant from the ion conduction path. These membrane proteins consist of distinct ligand-binding and pore domains that interact via an extended interface. Here, we have investigated the role of residues at this interface for channel activation to define critical interactions that couple conformational changes between the two structural units. By characterizing point mutants of the prokaryotic channels ELIC and GLIC by electrophysiology, X-ray crystallography and isothermal titration calorimetry, we have identified conserved residues that, upon mutation, apparently prevent activation but not ligand binding. The positions of nonactivating mutants cluster at a loop within the extracellular domain connecting β-strands 6 and 7 and at a loop joining the pore-forming helix M2 with M3 where they contribute to a densely packed core of the protein. An ionic interaction in the extracellular domain between the turn connecting β-strands 1 and 2 and a residue at the end of β-strand 10 stabilizes a state of the receptor with high affinity for agonists, whereas contacts of this turn to a conserved proline residue in the M2-M3 loop appear to be less important than previously anticipated. When mapping residues with strong functional phenotype on different channel structures, mutual distances are closer in conducting than in nonconducting conformations, consistent with a potential role of contacts in the stabilization of the open state. Our study has revealed a pattern of interactions that are crucial for the relay of conformational changes from the extracellular domain to the pore region of prokaryotic pentameric ligand-gated ion channels. Due to the strong conservation of the interface, these results are relevant for the entire family.

A chimeric prokaryotic-eukaryotic pentameric ligand gated ion channel reveals interactions between the extracellular and transmembrane domains shape neurosteroid modulation

“…Regardless of whether a new proton sensor is created or proton sensitivity of an existing site is altered, the biphasic proton dose response curves stem from mismatched interactions between the GLIC ECD and GABAρ TMD. These data provide support for the idea that agonist-mediated pLGIC gating transitions are regulated by specific interdomain interactions between the ECD and TMD (Bertozzi et al, 2016; Gupta et al, 2017; Lee and Sine, 2005; Xiu et al, 2005). …”

“…At the ECD-TMD interface, connections between flexible loops in the extracellular binding domain (loops 2, 7, 9) with the transmembrane channel domain (M2–M3 loop) structurally link the two domains and are essential for coupling ligand binding to channel gating (Miller and Smart, 2010). Agonist-mediated closed to open channel gating transitions are accompanied by substantial rearrangements of this interface (Bertozzi et al, 2016; Dellisanti et al, 2013; Gupta et al, 2017; Lee and Sine, 2005; Velisetty et al, 2014; Xiu et al, 2005). In chimeric channels assembled by combining the ECD and TMD of two distinct pLGICs, substantial loop substitutions are required to maintain complementarity and ensure normal channel function (Bouzat et al, 2008; Bouzat et al, 2004; Eisele et al, 1993).…”

Mutational Analysis at Intersubunit Interfaces of an Anionic Glutamate Receptor Reveals a Key Interaction Important for Channel Gating by Ivermectin

“…Previous functional studies with various Cys-loop receptor mutants have shown that the M2-M3 loop is involved in the gating process (Campos-Caro et al, 1996; Lynch et al, 1997; Boileau and Czajkowski, 1999; Grosman et al, 2000; Bera et al, 2002; Absalom et al, 2003; Kash et al, 2003; Bouzat et al, 2004, 2008; Grutter et al, 2005; Law et al, 2005; Lee and Sine, 2005; Lummis et al, 2005; Reeves et al, 2005; Sala et al, 2005; Xiu et al, 2005; Jansen and Akabas, 2006; Jha et al, 2007; Lee et al, 2008, 2009; Chang et al, 2009; Paulsen et al, 2009; Perkins et al, 2009; Pless and Lynch, 2009b; Wiltfong and Jansen, 2009; Yamodo et al, 2010; Hamouda et al, 2011; Zhang et al, 2011, 2013; Dellisanti et al, 2013; Mnatsakanyan and Jansen, 2013; Scott et al, 2015; Bertozzi et al, 2016). Hence, in this study we refrained from substituting amino acids in the M2-M3 loop or amino acids that, according to the X-ray crystal structure of the GluClα cryst R, might directly contribute to the bond network of the β1β2, Cys and β8β9 loops with the M2-M3 loop (Hibbs and Gouaux, 2011).…”

Photocontrol of endogenous glycine receptors in vivo

“…The compound displayed low activity and no photoswitching in 5-HT 3A R (Figure 2cand Supplementary Figure 7) and was completely inactive in muscular nAChR (Supplementary Figure 8). Glyght binds preferentially at a non-glycine site located at the interface of the extracellular and transmembrane domains (ECD and TMD) that is involved in the allosteric coupling between ligand binding to the ECD and opening 12 of the ion channel pore in the TMD [44][45][46][47][48] .e, Detailed view of the intersubunit site at the ECD/TMD interface including the protein loops changing conformation upon receptor activation 49 Figure 9-10). We focused on the latter binding site, since it is the region showing the largest differences in ligand pose densities for both α 1 Ζ and α 2 H GlyRs.…”

“…We focused on the latter binding site, since it is the region showing the largest differences in ligand pose densities for both α 1 Ζ and α 2 H GlyRs. Moreover, this site includes key residues for channel activation and conductance 50,51 and is involved in allosteric coupling between ligand binding to the ECD and ion channel pore opening in the TMD [44][45][46][47][48] . As shown in We have used a zebrafish behavioral assay and photoresponse score to identify the first GlyR-selective, light-regulated inhibitor.…”