Periodic Reporting for period 1 - ICaRO (Ion Channels and Receptors Operation)
Reporting period: 2016-06-13 to 2018-06-12
The physiological importance of ion channels is underlined by their involvement in a wide range of pathologies spanning all major therapeutic areas. Approximately 13% of known drugs have their primary therapeutic action at ion channels, making them the second largest target class after G-protein coupled receptors. However, our understanding of ion channel operation is limited to inferences based on functional data and to static snapshots of their structures where they are available. Cyclic nucleotide-modulated channels and calcium-activated K+ channels play crucial roles in a myriad of physiological processes ranging from signal transduction to neuronal excitability. The binding of ligands to specialized intracellular domains modulates the opening/closing (gating) equilibrium of these channels. Both the high-resolution structures - in open and closed conformation - of these channels and the precise mechanism of ligand-mediated channel activation are unknown. The present proposal aims to understand the mechanism of ion channel modulation by ligands. To accomplish this goal we utilized prokaryotic homologues of these channels reconstituted in artificial membranes. High-speed atomic force microscopy (HS-AFM) allowed us to directly observe individual ion channel molecules in action at high spatiotemporal resolution and in physiological conditions. Differences between liganded and unliganded channel structures provided us insight into the mechanism of ligand gating. The obtained data were compared to single channel current recordings obtained under comparable conditions.
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.
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.
The present proposal significantly advances our understanding of ion channels operation.
Understanding membrane proteins in their own environment, is still a major challenge of structural biology, not only because the determination of their 3D structure is still hard and demanding, but also because the gold standard techniques in the field, X-ray crystallography and single particle cryo-EM, provide the structure in physiochemical conditions that can be dramatically different from those occurring in nature. The project allowed to overcome these major limitations currently existing in the field. We were able to characterize the dynamic trajectory of a model ion channel functioning in close-to-physiological conditions (i.e. ambient temperature, liquid, and in a biological membrane) and at single molecule level. This achievement advances our mechanistic understanding of ligand gated ion channel operation and the technique shall be applicable to many other membrane proteins, and therefore be of broad relevance. Specifically, the increasing understanding of ligand-mediated ion channels structure-function relationship will allow the development of novel therapeutic agents for numerous disorders, including hypertension, epilepsy, and arrhythmia. Moreover, the technique pave the way to the 3D structure visualization of membrane proteins and their spatial organization in humane samples (biopsy). This will potentially explain why some mutations have no impact on a function of a protein, while others lead to clinical phenotype.
Understanding membrane proteins in their own environment, is still a major challenge of structural biology, not only because the determination of their 3D structure is still hard and demanding, but also because the gold standard techniques in the field, X-ray crystallography and single particle cryo-EM, provide the structure in physiochemical conditions that can be dramatically different from those occurring in nature. The project allowed to overcome these major limitations currently existing in the field. We were able to characterize the dynamic trajectory of a model ion channel functioning in close-to-physiological conditions (i.e. ambient temperature, liquid, and in a biological membrane) and at single molecule level. This achievement advances our mechanistic understanding of ligand gated ion channel operation and the technique shall be applicable to many other membrane proteins, and therefore be of broad relevance. Specifically, the increasing understanding of ligand-mediated ion channels structure-function relationship will allow the development of novel therapeutic agents for numerous disorders, including hypertension, epilepsy, and arrhythmia. Moreover, the technique pave the way to the 3D structure visualization of membrane proteins and their spatial organization in humane samples (biopsy). This will potentially explain why some mutations have no impact on a function of a protein, while others lead to clinical phenotype.