Conformational dynamics of cationic cys-loop receptors
Understanding the functional dynamics of ligand-gated ion-channels (LGIC's) on the molecular level is the aim of this thesis. Members of the protein family of LGIC's are involved in the fast synaptic signal transmission and thereby play a central role in inter-cellular communication in higher multi-cellular organisms. Here, two prototypical members of ligand-gated cys-loop receptors were studied: the homopentameric type 3 serotonin receptor (5HT3AR) and the muscle-type nicotinic acetylcholine receptor (nAchR). The single-channel kinetics of the 5HT3AR was investigated using single-channel patch-clamp and single-molecule fluorescence techniques. Our principal findings are that the 5HT3AR channel shows up to five, agonist activated, nearly equidistant conductance levels, by binding of up to five agonist molecules. Detailed analysis of data obtained using serotonin the native agonist and quipazine-TMR, a fluorescent partial agonist at different ionic conditions has been performed. An allosteric calcium modulation was observed with serotonin but not with the partial agonist indicating receptor activation with altered conformational pathways. In conclusion, a functional model is proposed, describing a two-way conformational activation. Binding of each agonist opens a "conductance filter" within the addressed subunit. Additionally, a global gate, which can be modulated by the presence of calcium ions, determines the channel kinetics. In order to identify at least one of the hypothesized conformational pathways of the 5HT3A receptor activation, a mutation of aspartate to cysteine at the position 298 was introduced into the receptor variant devoid of cysteine residues. Labelling the mutated residue with a fluorescent dye resulted in a fluorescence quenching upon receptor activation. This effect was interpreted as a functional conformational rearrangement. Although no specific time-scale of such a (local or global) change in conformation could be determined on the molecular level, the application of the described approach as a possible test for functionality upon receptor solubilisation is discussed. The simultaneous observation of the discrete ligand binding and the subsequent single-channel gating was discussed for years in the LGIC field as the method of choice for direct and unbiased examination of the channel activation process. Here, current recordings of single-channel gating events were combined with optical single-molecule detection of fluorescent agonist binding. The time delay Δt between ligand binding and gating events of a gain-of-function variant of the foetal and the wild-type adult nAchR were measured on the single molecule level on living cells. For times above approximately 1 ms, the distribution of Δt for both receptor types obeys a power-law with an exponent close to 3/2, corresponding to a normal conformational diffusion. Increasing the time resolution, a monoexponential function describes the obtained data best for times below 1 ms, suggesting that an activation barrier in the free energy landscape of the receptor activation can be overcome within one step. Finally, a novel method for the fast and simple induction of synapse formation was developed. Optical tweezers were used for pulling membrane-nanotubes out of the plasma membrane of a living cell. Connecting these nanotubes to the cell membrane of up to 100 µm distant cells, axonal or dendritic protrusions can be mimicked. Functional electrical synapses, based on gap junctions, were artificially created. The results obtained in this work contribute to the functional characterisation of LGIC's in living cells, yielding models that might be extrapolated as concepts of conformational dynamics of proteins in general.
Keywords: ligand gated ion channels ; cys-loop receptors ; acetylcholine receptor ; serotonin receptor ; protein dynamics ; artificial synapse ; patch-clamp ; confocal imaging ; optical tweezers ; fluorescence ; single moleculeThèse École polytechnique fédérale de Lausanne EPFL, n° 4524 (2009)
Programme doctoral Chimie et Génie chimique
Faculté des sciences de base
Institut des sciences et ingénierie chimiques
Laboratoire de chimie physique des polymères et membranes
Record created on 2009-09-03, modified on 2016-08-08