The overall objective of the proposal was to understand the mechanism of ion channel modulation by ligands. To accomplish this goal we used prokaryotic homologues of Ca²⁺-activated K⁺ channels and cyclic nucleotide-gated channels reconstituted in artificial membranes. These preparations were then studied by high-speed atomic force microscopy (HS-AFM) wich allowed us to directly observe individual ion channel molecules in action at high spatiotemporal resolution and in physiological conditions. As a model system for the first class of ion channels, the bacterial MthK channel was selected. Unfortunately we could not succeed from this preparation in obtaining high-quality topographies (mainly due to the fast lateral diffusion of the protein which impeded high-resolution imaging). As a model system for the second class of ion channels, the bacterial SthK channel was selected. From this sample we got high resolution images in the presence of the ligand (cAMP), in it's absence and in the presence of a competitive antagonist (cGMP). We also obtained information about the dynamics of the observed conformational change in response to ligand; during imaging, cAMP or cGMP was delivered by a high-precision pumping system and the conformtional change monitored. From the data we collected, we could conclude that upon ligand binding conformational changes are not limited to the ligand binding pocket, but spreads to channels perihery, resulting in a reorganization of the whole crystal lattice and involving major rearrangment in the voltage sensing domain, where most of the crystal contacts take place. This data demonstrate the existance of long-range interactions between different and distant protein domains previously overlooked.
The results were presented at several international confernces (EMBO Molecular Neurobiology, Crete, 2018; RECI VI Meeting, Santiago, 2017; Biophysical Society 62nd Annual Meeting, San Francisco, 2018) and are currently published or under revision in top tier scientifc journals.
