SFB Transregio ReceptorLight

High-end light microscopy elucidates membrane receptor function

Following the binding of ligands, membrane receptors generate signals that control the cells of an organism in a multifaceted manner. In the past years new light microscopy methods have provided essentially new insights into the function of membrane receptors, for example into the rates of ligand binding to the receptors and the subsequent conformational changes. Concerning the localization of the receptors, a spatial resolution in the range of 20 nm has been reached, which is far below the optical diffraction limit reported by Ernst Abbe.

In the CRC/TR ReceptorLight high-end light microscopy techniques with highest spatial and time resolution were applied and further developed to gain deeper insight into the function of membrane receptors. The working groups in Jena and Würzburg contributing to ReceptorLight have bundled their methodological expertise in the field of high-end microscopy with that in the fields of physiology and biophysics of membrane receptors. The aim of this collaboration was to gain new insight into the function and distribution of diverse membrane receptors, and in parallel, to induce the development of new high-end light-microscopy methods. The 21 projects used e.g. super-resolution microscopy, 3-dimensional two photon calcium imaging, single-molecule strategies, tip-enhanced Raman spectroscopy, confocal patch-clamp fluorometry, Förster resonance energy transfer analyses and fluorescence correlation spectroscopy as well as combinations thereof. These methods and complex mathematical algorithms for the analyses of the data were used by the participants of ReceptorLight in close collaboration.

The following projects were run at the Institute of Physiologie II:

A5 Relating the binding of single ligands to single HCN and CNG channels

From single cyclic nucleotide-gated channels (CNGA2) the binding of single labelled cGMP molecules and the single-channel activity will be simultaneously recorded. In addition, a single-molecule approach will be developed for supported membranes to quantify the dwell time of single labelled cAMP molecules at the four cyclic-nucleotide binding domains of individual homotetrameric HCN2 channels. Novel cGMP and cAMP derivatives will be synthesized. The results should help to unravel the interaction of the four subunits in CNGA2 and HCN2 channels and how this interaction is controlled by voltage in HCN2 channels.

Prof. Dr. Klaus Benndorf

Phone: 03641 - 9 397651

B1 Elucidating assembly, ligand binding and gating of heterotetrameric CNG channels by FRET

Cyclic nucleotide-gated (CNG) channels play an important role in the visual and olfactory system. They constitute the final step of a signal transduction cascade by which light or scent are transduced into a change of the membrane potential. Native CNG channels are heterotetramers, consisting of up to three different subunits. Each subunit has six membrane-spanning segments. The binding of a cyclic nucleotide (cAMP or cGMP) to the cyclic nucleotide binding domains (CNBDs) located intracellularly at the C-terminus promotes an allosteric conformational change that is transmitted through the C-linker to the channel pore leading to channel opening.

Although all subunits contain an intracellular located cyclic nucleotide-binding domain, only the CNGA1, CNGA2 and CNGA3 are capable of forming functional homotetrameric channels when expressed in heterologous expression systems. CNGA4 and CNGB subunits only modulate channel function and control plasma membrane targeting. Direct evidence for their contribution to channel activation by ligand binding was presented up to now only for the olfactory CNGA4 subunit.

This project aims at elucidating the role of the CNGB1a and CNGB3 subunits of our visual system. Specifically, we want to find out whether these subunits bind cGMP at physiological concentrations and if so, in which sequence binding to the respective subunits occurs.

The following questions will be addressed: In which sequence do the ligands bind to the subunits? Is the opening of the channel pore the result of a concerted conformational change of all four subunits? What conformational changes are induced by ligand binding? To address these questions we will use among other methods confocal patch-clamp fluorometry (cPCF) and Förster Resonance Energy Transfer (FRET) measurements.

A second focus of the project will be on the mechanisms by which the cell ensures that channels are expressed in a fixed stoichiometry. These mechanisms are responsible for the successful channel trafficking to the plasma membrane whose malfunction was shown to be the cause of many channelopathies. By using fluorescence lifetime based FRET measurements we will investigate in which compartment the channels assemble, which subunits associate first, and how channel transport is controlled.

Dr. Vasilica Nache

Phone: 03641 - 9 397668

B7 Relating ligand binding and activation gating in nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) belong to the superfamily of cys-loop neurotransmitter-gated ion channels. They are expressed in different neuronal and non-neuronal cell types throughout the body, where they mediate fast synaptic cholinergic transmission. nAChR current responses are characterized by a rapid activation upon acetylcholine binding followed by a complex desensitization in the presence of the agonist. While the activation behavior is well understood, the desensitization is still a matter of debate. This project is intended to contribute to the understanding of the desensitization process in adult muscle-type nAChRs.

Because desensitized states are high-affinity states, which are electrically silent, the technical focus will be on the confocal patch-clamp fluorometry (cPCF), an approach combining electrophysiological patch-clamp techniques with confocal fluorescence microscopy. Using a fluorescently tagged acetylcholine analogue, this approach allows not only to control and to monitor receptor activation but also to simultaneously read out ligand binding. In contrast to pure electrophysiological approaches, state-dependent ligand binding can be monitored directly, thus contributing to a more complete picture of the desensitization process.

Dr. Jana Kusch

Phone: 03641 - 9 397668

C4 Kinetics of metabotropic glutamate receptors

The activation time among G protein-coupled receptors (GPCRs) seems to vary by more than five orders of magnitude, whereupon methodological reasons severely limit any analysis. Optimized systems will be developed to accurately measure the activation kinetics of metabotropic GPCRs, including light-induced activation by concentration jumps evoked by either caged agonists or a piezo device as well as photosensitive tethered agonists. The readout for receptor activation will be Förster Resonance Energy Transfer from receptor constructs with appropriate fluorophores. The data will be analyzed by Markovian models.

Prof. Dr. Klaus Benndorf

Phone: 03641 - 9 397651