Single-molecule spectroscopy of the β2 adrenergic receptor: Observation of conformational substates in a membrane protein

Single-molecule studies of the conformations of the intact β2 adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the β2 adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.

Quantification of spread of cerebellar long-term depression with chemical two-photon uncaging of glutamate

Localized, chemical two-photon photolysis of caged glutamate was used to map the changes in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors caused by long-term synaptic depression (LTD) in cerebellar Purkinje cells. LTD produced by pairing parallel fiber activity with depolarization was accompanied by a decline in the response of Purkinje cells to uncaged glutamate that accounted for both the time course and magnitude of LTD. This depression of glutamate responses was observed not only at the site of parallel fiber stimulation but also at more distant sites. The amount of LTD decreased with distance and was half-maximal 50 μm away from the site of parallel fiber activity. Estimation of the number of parallel fibers active during LTD induction indicates that LTD modified glutamate receptors not only at active synapses but also at 600 times as many inactive synapses on a single Purkinje cell. Therefore, both active and inactive parallel fiber synapses can undergo changes at a postsynaptic locus as a result of associative pre- and postsynaptic activity.