Kinetic study of two pentameric ligand-gated ion channels

The Cys-loop or nicotinic superfamily is an important group of ligand-gated ion channels. The pentameric channels in this family commonly mediate fast synaptic transmission in eukaryotes (cf. nicotinic acetylcholine, γ-aminobutyric acid receptor and glycine receptor). Related channels are also found in prokaryotes and these channels include Gloeobacter violaceous ligand-gated ion channel (GLIC) and Erwinia chrysanthemi ligand gated ion channel (ELIC). Cys-loop channels share a common structural fold and are thought to activate in a similar way: the ligand binds at the interface between the extracellular domains of adjacent subunits and causes the receptor to undergo a conformational change. This is conveyed to the transmembrane region of the receptor where it results in the opening of the channel pore. The energy landscape of the channel protein can be characterised by determining a kinetic mechanism, which explicitly details the functional states that the channel can visit and quantifies the transition rates between them. This is possible in ion channels because the current generated by a single channel molecule can be detected with high temporal resolution. Establishing a detailed kinetic mechanism allows a better understanding of the structure function relation for the protein, because it allows us to determine which step of the activation is affected when we change agonist or when we mutate the channel protein itself. The aim of my work was to establish the kinetic mechanism that best describes two homomeric ligand-gated ion channels, the α3 glycine receptor and ELIC receptor. This work involved recording single channel currents and the currents elicited by fast agonist applications and analysis by direct model fitting to the data. The physiological role of α3 glycine receptor is not currently known. The α3 expression is concentrated in areas involved in pain processing, such as the superficial dorsal horn (Harvey et al., 2004), suggesting that this isoform could be involved in the nociceptive pathway. Characterising the kinetics of a synaptic receptor is important also in understanding synaptic transmission, because it is the lifetime of the activated channel that is largely responsible for setting the time course of synaptic currents. We investigated the activation mechanism of this channel in HEK293 cells by maximum likelihood fitting of single-channel data, at a wide range of glycine concentrations. The mechanism we propose suggests that α3 channels can open only when more than 3 binding sites are occupied by glycine, and only after the channel undergoes a conformational change (‘flip’) that links binding to gating. The scheme can describe adequately macroscopic currents from fast concentration jumps experiments. The function of the prokaryotic channel ELIC is unknown, but ELIC is important as it has been recently crystallized (Hilf and Dutzler, 2008) in non-conductive state (the highest resolution structure currently available for a non-conductive state). Establishing a kinetic mechanism for this channel is particularly important, because combining structure and function offers the best possibility to investigate how the perturbation induced by the binding of the transmitter opens the channel. We investigated the activation mechanism of this channel in HEK293 cells by maximum likelihood fitting of single-channel data, at a wide range of propylamine concentrations. The mechanism we propose suggests that ELIC can open from partially primed conformational states, and only after the channel undergoes a conformational change (‘priming’), which links binding to gating. The scheme describes adequately both macroscopic currents from fast concentration jumps experiments and single channel activity.