Abstract
Electrochemical signaling in the brain depends on pentameric ligand-gated ion channels (pLGICs). Recently, crystal structures of prokaryotic pLGIC homologues from Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus (GLIC) in presumed closed and open channel states have been solved, which provide insight into the structural mechanisms underlying channel activation. Although structural studies involving both ELIC and GLIC have become numerous, thorough functional characterizations of these channels are still needed to establish a reliable foundation for comparing kinetic properties. Here, we examined the kinetics of ELIC and GLIC current activation, desensitization, and deactivation and compared them to the GABAA receptor, a prototypic eukaryotic pLGIC. Outside-out patch-clamp recordings were performed with HEK-293T cells expressing ELIC, GLIC, or α1β2γ2L GABAA receptors, and ultra-fast ligand application was used. In response to saturating agonist concentrations, we found both ELIC and GLIC current activation were two to three orders of magnitude slower than GABAA receptor current activation. The prokaryotic channels also had slower current desensitization on a timescale of seconds. ELIC and GLIC current deactivation following 25 s pulses of agonist (cysteamine and pH 4.0 buffer, respectively) were relatively fast with time constants of 24.9±5.1 ms and 1.2±0.2 ms, respectively. Surprisingly, ELIC currents evoked by GABA activated very slowly with a time constant of 1.3±0.3 s and deactivated even slower with a time constant of 4.6±1.2 s. We conclude that the prokaryotic pLGICs undergo similar agonist-mediated gating transitions to open and desensitized states as eukaryotic pLGICs, supporting their use as experimental models. Their uncharacteristic slow activation, slow desensitization and rapid deactivation time courses are likely due to differences in specific structural elements, whose future identification may help uncover mechanisms underlying pLGIC gating transitions.
Highlights
Pentameric ligand gated ion channels mediate excitatory and inhibitory synaptic transmission and their evolutionary precursors have been identified in several bacteria [1]
We examined currents evoked by GABA, cysteamine, and changes in pH from the GABAAR, ELIC, and Gloeobacter violaceus (GLIC), respectively, expressed in X. laevis oocytes (Figure 1A)
GABA macroscopic kinetics When 10 mM GABA is applied to a patch for 1 s, the macroscopic kinetics of the GABAAR current has three distinct phases: a fast rising current activation phase with a time constant (t-activation) of 0.8 ms60.1 ms, n = 19, a desensitization phase, where current decreases in the presence of agonist, with a weighted time constant of 162637 ms, n = 10, and a deactivation phase, where current decays back to baseline after removal of GABA, with a weighted time constant of 10468 ms, n = 10 (Figure 2A)
Summary
Pentameric ligand gated ion channels (pLGICs) mediate excitatory and inhibitory synaptic transmission and their evolutionary precursors have been identified in several bacteria [1]. These channels, comprised of five homologous subunits arranged pseudo-symmetrically around a central ion conducting pore, are structurally adapted to rapidly convert chemical signals (i.e. the binding of ligands) into electrical signals (i.e. ion flow through a central pore). Two prokaryotic pLGIC homologues have been identified, ELIC and GLIC, from the plant pathogen Erwinia chrysanthemi and the cyanobacterium Gloeobacter violaceus, respectively [1,2]. ELIC has been crystallized in a presumed closed state [4] and GLIC in an apparent open state [5,6]. Comparison of the structures reveals distinct conformational changes in the extracellular-binding domain and transmembrane domain that have been used to predict closed to open pLGIC gating mechanisms
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