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Title: Ligand Activation of the Prokaryotic Pentameric Ligand-Gated Ion Channel ELIC}
Article:
Abstract:
While the pentameric ligand-gated ion channel ELIC has recently provided first insight into the architecture of the family at high resolution, its detailed investigation was so far prevented by the fact that activating ligands were unknown. Here we describe a study on the functional characterization of ELIC by electrophysiology and X-ray crystallography. ELIC is activated by a class of primary amines that include the neurotransmitter GABA at high micro- to millimolar concentrations. The ligands bind to a conserved site and evoke currents that slowly desensitize over time. The protein forms cation selective channels with properties that resemble the nicotinic acetylcholine receptor. The high single channel conductance and the comparably simple functional behavior make ELIC an attractive model system to study general mechanisms of ion conduction and gating in this important family of neurotransmitter receptors.
Introduction:
The pentameric ligand-gated ion channels( pLGICs) constitute a large family of ionotropic neurotransmitter receptors that are ubiquitously expressed in the animal kingdom. In vertebrates the family encompasses cation selective serotonin and nicotinic acetylcholine receptors( nAChRs) and anion selective GABA and glycine receptors[1]. All family members share a conserved molecular architecture and a similar functional behavior. The proteins act as gated ion channels that are closed in their resting state and that open a selective ion conduction pore upon the binding of a ligand to a site located on an extracellular domain[2]–[6]. These properties are shared by close prokaryotic homologues that are encoded in the genome of certain bacterial species[7]–[9]. The X-ray structures of two prokaryotic pLGICs from the plant pathogen Erwinia chrysanthemi( or Dickeya dadantii, ELIC)[10] and the cyanobacterium Gloebacter violaceous( GLIC)[11],[12] have recently revealed first structural insight into the family at high resolution. While both structures are similar overall, distinct conformational differences in the ligand binding domain and the transmembrane pore domain indicate that they represent different functional states of the protein, with ELIC showing a non-conducting and GLIC a conducting state( Figure S1). The functional properties of GLIC, which is opened by a decrease of the extracellular pH, have been investigated by electrophysiology[13]. The protein forms cation selective channels with slow opening kinetics and currents that are stable over an extended period of time. The latter property differs from most eukaryotic pLGICs that quickly inactivate after opening[14]–[16]. The analogous ion permeation properties and the sensitivity to the same set of open channel blockers underline the close structural relationship of the ion conduction pore between GLIC and the nAChR[13],[17], which is also manifested in the sequence conservation of this region( Figure S1G). Although, based on sequence similarity and an initial functional characterization[10], ELIC is also expected to belong to the same group of cation selective channels, it has so far been impossible to investigate its detailed properties since the stimulus for its activation was unknown. ELIC shows a non-conducting conformation of the pore[10],[18], whose correspondence to the resting state of eukaryotic pLGICs is currently unclear[7],[19]. The structural similarity of its extracellular part to the acetycholine binding protein( AChBP), a soluble protein that closely resembles the ligand-binding domain of nAChRs, however, suggests a conserved mode for ligand interactions( Figure 1, Figure S2)[10],[20]. Structures of the AChBP in complex with different agonists of the nAChR have provided a detailed picture of this process and have helped to rationalize previous biochemical studies[3],[21]–[23]. The ligand binding site is placed in a pocket at the interface of adjacent subunits of the pentameric protein. The two halves of the pocket are termed “principal” and “complementary side, ” respectively, depending on the subunit in which the contributing residues are located. On each subunit residues from different regions of the molecule line the ligand binding pocket. Their location was named according to early experiments( Figure S2A)[24]. Based on this nomenclature the principal side contains the regions A, B, and C and the complementary site the regions D, E, and F. The conserved residues include several aromatic side chains, which provide delocalized electrons to stabilize the positively charged quaternary ammonium group( Figure S2C)[25],[26]. The structural similarity between ELIC and AChBP extends to the ligand binding site, which also in ELIC contains conserved aromatic positions that surround a pocket of about the same volume( Figure 1, Figure S2). It was thus assumed that ELIC might be opened by a ligand of similar size and chemical properties[10]. Here we have investigated the activation of ELIC by small molecular ligands by X-ray crystallography and different electrophysiological techniques. Our study has identified a class of primary amines that open the channel by a strongly cooperative process. Although the open state is comparably stable the channels desensitize with slow kinetics. An X-ray structure of ELIC in complex with a ligand shows electron density in the consensus binding site, thus suggesting that the mechanism of activation is conserved. The open channel of ELIC mediates the permeation of cations with a large single channel conductance and selectivity properties that resemble other cation selective members of the family.
Discussion:
Although the prokaryotic pLGIC ELIC has provided the first structural insight into this family of ligand-gated ion channels at high resolution, its functional characterization was so far prevented by the fact that activating ligands were unknown. In this study we have identified agonists of ELIC from a library of molecules that were selected based on the chemical features of the ligand binding site, we have located a bound ligand in a crystal structure, and we have characterized the functional properties of the channel by electrophysiology. ELIC is activated by a set of primary amines that include alkylamines, aminothiols, aminoalcohols, bromoamines, diamines, and the neurotransmitter GABA. Quaternary amines, such as acetylcholine, in contrast, are ineffective. The chemical properties of the agonists underline the conservation of the ligand binding pocket that is closer to GABA and glycine receptors than to nAChRs and AChBPs( Figure S5). As is the case for other pLGICs[41],[42], ligand activation in ELIC is a cooperative process and appears to involve the binding of at least three molecules to maximally activate the protein. However, unlike its eukaryotic counterparts[43], all agonists activate ELIC with comparably low apparent affinity at high µM to mM concentrations. While it is still not known whether the ligand activating ELIC in a biological context is among the characterized molecules, it is noteworthy that several of the agonists are breakdown products of amino acids, two of which, putrescine and GABA, may play a role in the natural environment of the bacteria, which as pathogens degrade the roots of plants. It may thus not be a coincidence to find the gene of a putative amino acid decarboxylase in the direct vicinity of the gene encoding for ELIC on the bacterial chromosome( Figure 6A). This protein, which might be involved in the processing of the ligand, was annotated as glutamate decarboxylase, thus suggesting that, despite its low potency, GABA is among the likely candidates for natural ligands. The macroscopic opening and closing kinetics of ELIC is in the millisecond time range. Although the rates are slow compared to most eukaryotic family members[44],[45], they are similar to the previously investigated activation and deactivation rates of GLIC[13]. In contrast to most eukaryotic pLGICs[15], the evoked currents are stable for several hundred milliseconds, but they slowly decay, due to the transition into a desensitized state. This behavior is consistent with long periods of closures separating bursts of channel openings upon prolonged exposure to saturating concentrations of the ligand observed in single channel traces. Within the bursts, the open probability of the channel is high, which is indicative for a high efficacy of the investigated ligands[32],. The detailed kinetic characterization of gating based on single channel recordings will be the subject of future investigations. It is currently still unclear whether the crystal structure of ELIC, which shows a non-conducting conformation of the channel obtained in the absence of ligands, is closer to the resting or the desensitized state of the channel. Consequently, its relationship to the resting state of eukaryotic receptors is also unknown[19],[47]. A previous hypothesis that the observed conformation would be induced by strong interactions of aromatic phenylalanine residues at the extracellular pore entry[31], however, could not be substantiated since the mutation to alanine did not change the overall structure and instead stabilized the closed state of the channel as indicated by a decrease in the potency of the agonist. This observation is unexpected, as hydrophobic interactions generally play an important role in the stabilization of protein structures. In an allosteric model of channel activation, the binding of the ligand shifts the equilibrium between the open and closed channel towards the open state[48],[49]. For that purpose, the agonist has to bind with higher affinity to the open state of the channel. In this respect, it is interesting to compare the conformation of the ligand binding domain of ELIC with a hypothetical conformation of the domain in the open state that was generated by a rigid body transformation to the structure of GLIC( Figure 6B). In this hypothetical conformation, the binding site has contracted and is no longer accessible to the solvent. While the site is still large enough to accommodate the different ligands, their interaction with the protein would likely be stronger, thus fulfilling an important requirement for ligand activation. With respect to its ion conduction properties, ELIC is closely related to nAChRs, serotonin receptors, and GLIC[13],[34],[37],[50],[51]. ELIC is a cation selective channel that is essentially impermeable for anions. It does not discriminate between different monovalent cations and allows the permeation of divalent cations, which appear to interact stronger with the protein. The single channel conductance of ELIC is high; it is about 10 times higher than observed in GLIC[13], and it is comparable to the fastest conducting members of the family[39],[52],[53]. While a detailed understanding of this property will require more experimental data, it is still interesting to compare the pore geometry of GLIC and a hypothetic open state of ELIC that is based on the conformation of the pore forming helix observed in GLIC( Figure 6B). Despite the similarity of the protein backbone, the pore geometries of ELIC and GLIC would differ in such a model due to the different size of the side-chains of pore-lining residues. While the bulky Phe246 and Leu239 at the extracellular half of the pore of ELIC would result in a smaller pore diameter, the constricting intracellular diameter, which might be the main determinant for the conductance, could be larger( Figure 6B). Part of this larger pore diameter is caused by the location of the conserved Glu229 at the intracellular pore entry that is shifted by one residue in GLIC. With known agonists, ELIC has now become amenable for detailed structural and functional investigations. A fundamental question of ionotropic receptors concerns the mechanisms by which the binding of ligands induces channel activation over a large distance in a different part of the protein. ELIC is well suited to study the details of this process: Its large conductance will allow investigations of ion conductance and gating on a single channel level, which will additionally be facilitated by its comparably simple functional properties. Its high expression level and biochemical stability make it a suitable candidate for spectroscopy, and finally, knowledge of the ligand opens up the possibility to obtain structural information of a conducting conformation of the protein.
Electrical signal transduction at chemical synapses of nerve and muscle cells is initiated by the binding of neurotransmitters to their receptors , which function as gated ion channels that open a selective ion conduction path in the ligand-bound state . The pentameric ligand-gated ion channels constitute a very important family of neurotransmitter receptors that includes , amongst others , the nicotinic acetylcholine receptor . While most pLGICs are expressed in higher eukaryotes , there are few family members found in bacteria . These bacterial channels share the overall structural features of the family and are thus expected to function by similar mechanisms , but although the channel from the bacteria Erwinia chrysanthemi has revealed first structural insight into the family at high resolution , its functional characterization was impossible since activating ligands were not known . In our study we have identified ligands activating ELIC and we have located their binding by X-ray crystallography . Ligand binding causes the transition into an open state , which , as in eukaryotic receptors , is only transient and which slowly inactivates in the presence of the ligand . By single channel analysis we found that , similar to the acetylcholine receptor , ELIC forms cation selective channels . With known ligands ELIC has become an important model system to study the general mechanisms of the family .