Fluorescence Titrations to Determine the Binding Affinity of Cyclic Nucleotides to SthK Ion Channels

Background

Understanding the function of a protein in molecular detail requires extensive microscopic characterization. For ligand-gated ion channels different assays are needed to gain information about the specific interaction of the protein with the ligand, the communication between the ligand binding site and the pore, as well as channel-specific characteristics such as ionic throughput and inactivation or desensitization properties. Together with structural data for channel conformations corresponding to various functional states, this allows the development of a complete mechanistic description of channel function and regulation. Cyclic nucleotide-gated (CNG) ion channels are tetrameric potassium channels that are of particular interest due to their function in olfactory and visual signaling cascades (Kaupp and Seifert, 2002; Craven and Zagotta, 2006). However, there is only very limited data available on purified CNG channels under defined conditions, mostly from single molecule force spectroscopy (Higgins et al., 2002; Maity et al., 2015; Goldschen-Ohm et al., 2016; Mazzolini et al., 2018). Electrophysiological characterization of CNG channels expressed in Xenopus oocytes or mammalian cells (Biel et al., 1993 and 1994; Baumann et al., 1994; Weyand et al., 1994; Yu et al., 1996; Zagotta and Siegelbaum, 1996), shows that they are activated by micromolar concentrations of cyclic nucleotides (cNMP), but cAMP and cGMP can act differentially on different channel subtypes. High resolution structures of a few cyclic nucleotide-modulated channels have recently been solved (James et al., 2017; Lee and MacKinnon, 2017; Li et al., 2017; Rheinberger et al., 2018), highlighting the need for biophysical assays to characterize these channels in order to gain a better understanding of the molecular interactions during gating and regulation.

Recently, we introduced SthK, a bacterial homologue of eukaryotic CNG channels, as a model to analyze the allosteric regulation of these channels in vitro (Schmidpeter et al., 2018). One interesting observation was that cAMP and cGMP activate SthK with considerably different efficacies but interact with the protein with very similar affinities. We used fluorescently labeled cyclic nucleotides (Figure 1) to measure the binding affinity of these ligands to full-length SthK channels. The fluorescence of the NBD moieties increases upon interaction with a binding partner and thus directly reports the complex formation with SthK. To probe direct binding of unlabeled nucleotides to the channel we employed fluorescence-based competition assays. In the following we will provide detailed protocols for both experiments which can be performed in any protein-biochemistry laboratory using standard equipment and are applicable to protein-ligand interactions in general. Furthermore, this assay can be adapted to a plate-reader format which might increase the throughput and at the same time account for possible pipetting errors.


Figure 1. Structural formula of fluorescently labeled cyclic nucleotides. A. 8-NBD-cAMP. B. 8-NBD-cGMP. For both compounds the fluorescent group is labelled separately, the exact chemical name, the abbreviation and the trivial name are given. The representations were adapted from Biolog (Bremen, Germany, www.biolog.de).