Proton transfer unlocks inactivation in cyclic nucleotide-gated A1 channels

Key points: Desensitization and inactivation provide a form of short-term memory controlling the firing patterns of excitable cells and adaptation in sensory systems. Unlike many of their cousin K+ channels, cyclic nucleotide-gated (CNG) channels are thought not to desensitize or inactivate. Here we report that CNG channels do inactivate and that inactivation is controlled by extracellular protons. Titration of a glutamate residue within the selectivity filter destabilizes the pore architecture, which collapses towards a non-conductive, inactivated state in a process reminiscent of the usual C-type inactivation observed in many K+ channels. These results indicate that inactivation in CNG channels represents a regulatory mechanism that has been neglected thus far, with possible implications in several physiological processes ranging from signal transduction to growth cone navigation. Ion channels control ionic fluxes across biological membranes by residing in any of three functionally distinct states: deactivated (closed), activated (open) or inactivated (closed). Unlike many of their cousin K+ channels, cyclic nucleotide-gated (CNG) channels do not desensitize or inactivate. Using patch recording techniques, we show that when extracellular pH (pHo) is decreased from 7.4 to 6 or lower, wild-type CNGA1 channels inactivate in a voltage-dependent manner. pHo titration experiments show that at pHo < 7 the I-V relationships are outwardly rectifying and that inactivation is coupled to current rectification. Single-channel recordings indicate that a fast mechanism of proton blockage underlines current rectification while inactivation arises from conformational changes downstream from protonation. Furthermore, mutagenesis and ionic substitution experiments highlight the role of the selectivity filter in current decline, suggesting analogies with the C-type inactivation observed in K+ channels. Analysis with Markovian models indicates that the non-independent binding of two protons within the transmembrane electrical field explains both the voltage-dependent blockage and the inactivation. Low pH, by inhibiting the CNGA1 channels in a state-dependent manner, may represent an unrecognized endogenous signal regulating CNG physiological functions in diverse tissues

Unfolding and identification of membranproteins in situ

Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscope (AFM) to apply a force able to unfold a single protein. The obtained force-distance curve encodes the unfolding pathway, and from its analysis it is possible to characterize the folded domains. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer. Here, we describe a pipeline for analyzing membrane proteins based on SMFS, which involves the isolation of the plasma membrane of single cells and the harvesting of force-distance curves directly from it. We characterized and identified the embedded membrane proteins combining, within a Bayesian framework, the information of the shape of the obtained curves, with the information from mass spectrometry and proteomic databases. The pipeline was tested with purified/reconstituted proteins and applied to five cell types where we classified the unfolding of their most abundant membrane proteins. We validated our pipeline by overexpressing four constructs, and this allowed us to gather structural insights of the identified proteins, revealing variable elements in the loop regions. Our results set the basis for the investigation of the unfolding of membrane proteins in situ, and for performing proteomics from a membrane fragment

The permeation mechanism of organic cations through a CNG mimic channel

Several channels, ranging from TRP receptors to Gap junctions, allow the exchange of small organic solute across cell membrane. However, very little is known about the molecular mechanism of their permeation. Cyclic Nucleotide Gated (CNG) channels, despite their homology with K+channels and in contrast with them, allow the passage of larger methylated and ethylated ammonium ions like dimethylammonium (DMA) and ethylammonium (EA). We combined electrophysiology and molecular dynamics simulations to examine how DMA interacts with the pore and permeates through it. Due to the presence of hydrophobic groups, DMA enters easily in the channel and, unlike the alkali cations, does not need to cross any barrier. We also show that while the crystal structure is consistent with the presence of a single DMA ion at full occupancy, the channel is able to conduct a sizable current of DMA ions only when two ions are present inside the channel. Moreover, the second DMA ion dramatically changes the free energy landscape, destabilizing the crystallographic binding site and lowering by almost 25 kJ/mol the binding affinity between DMA and the channel. Based on the results of the simulation the experimental electron density maps can be re-interpreted with the presence of a second ion at lower occupancy. In this mechanism the flexibility of the channel plays a key role, extending the classical multi-ion permeation paradigm in which conductance is enhanced by the plain interaction between the ions

History, rare and multiple events of mechanical unfolding of repeat proteins

International audienceMechanical unfolding of proteins consisting of repeat domains is an excellent tool to obtain large statistics. Force spectroscopy experiments using AFM on proteins presenting multiple domains have revealed that unfolding forces depend on the number of folded domains (history) and have reported intermediate states and rare events. However, the common use of unspecific attachment approaches to pull the protein of interest holds important limitations to study unfolding history and may lead to discarding rare and multiple probing events due to the presence of unspecific adhesion and uncertainty on the pulling site. Site-specific methods recently emerged minimize this uncertainty and would be excellent tools to probe unfolding history and rare events. However, detail characterization of these approaches is required to identify their advantages and limitations. Here, we characterize a site-specific binding approach based on the ultrastable complex dockerin/cohesin III revealing its advantages and limitations to assess the unfolding history and to investigate rare and multiple events during the unfolding of repeated domains. We show that this approach is more robust, reproducible and provide larger statistics than conventional unspecific methods. We show that the method is optimal to reveal the history of unfolding from the very first domain, and to detect rare events, while being more limited to assess intermediate states. Finally, we quantify the unfolding forces required to unfold two molecules pulled in parallel, difficult using unspecific approaches. The proposed method represents a step forward towards more reproducible measurements to probe protein unfolding history and opens the door to systematic probing of rare and multiple molecule unfolding mechanisms

High resolution and High-Speed Atomic Force Microscope imaging

International audienceThe advent of high-speed atomic force microscopy (HS-AFM) in recent years has opened up new horizons for the study of structure, function and dynamics of biological molecules. HS-AFM is capable of 1000 times faster imaging than conventional AFM. This circumstance uniquely enables the observation of the dynamics of all the molecules present in the imaging area. In the last ten years, the HS-AFM has gone from a prototype-state technology that only a few labs in the world had access to (including ours) to an established commercialized technology that is present in tens of labs around the world. In this protocol chapter we share with the readers our practical know-how on high resolution HS-AFM imaging

Simulation atomic force microscopy for atomic reconstruction of biomolecular structures from resolution-limited experimental images

none5si: Atomic force microscopy (AFM) can visualize the dynamics of single biomolecules under near-physiological conditions. However, the scanning tip probes only the molecular surface with limited resolution, missing details required to fully deduce functional mechanisms from imaging alone. To overcome such drawbacks, we developed a computational framework to reconstruct 3D atomistic structures from AFM surface scans, employing simulation AFM and automatized fitting to experimental images. We provide applications to AFM images ranging from single molecular machines, protein filaments, to large-scale assemblies of 2D protein lattices, and demonstrate how the obtained full atomistic information advances the molecular understanding beyond the original topographic AFM image. We show that simulation AFM further allows for quantitative molecular feature assignment within measured AFM topographies. Implementation of the developed methods into the versatile interactive interface of the BioAFMviewer software, freely available at www.bioafmviewer.com, presents the opportunity for the broad Bio-AFM community to employ the enormous amount of existing structural and modeling data to facilitate the interpretation of resolution-limited AFM images.noneAmyot, Romain; Marchesi, Arin; Franz, Clemens M; Casuso, Ignacio; Flechsig, HolgerAmyot, Romain; Marchesi, Arin; Franz, Clemens M; Casuso, Ignacio; Flechsig, Holge

An iris diaphragm mechanism to gate a cyclic nucleotide-gated ion channel

Cyclic nucleotide-gated (CNG) ion channels are non-selective cation channels key to signal transduction. The free energy difference of cyclic-nucleotide (cAMP/cGMP) binding/unbinding is translated into mechanical work to modulate the open/closed probability of the pore, i.e., gating. Despite the recent advances in structural determination of CNG channels, the conformational changes associated with gating remain unknown. Here we examine the conformational dynamics of a prokaryotic homolog of CNG channels, SthK, using high-speed atomic force microscopy (HS-AFM). HS-AFM of SthK in lipid bilayers shows that the CNBDs undergo dramatic conformational changes during the interconversion between the resting (apo and cGMP) and the activated (cAMP) states: the CNBDs approach the membrane and splay away from the 4-fold channel axis accompanied by a clockwise rotation with respect to the pore domain. We propose that these movements may be converted by the C-linker to pull the pore helices open in an iris diaphragm-like mechanism