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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.

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.

We provide the BioAFMviewer-Toolbox, an extension of our previously developed software platform for simulated AFM scanning of biomolecular structures and dynamics. The focus was on developing a toolbox of methods which employ simulated AFM scanning combined with quantitative analysis to facilitate the interpretation of resolution-limited AFM images. The key advancement is the automatized fitting of biomolecular structures to experimental AFM images, which allows to reconstruct 3D atomistic structures from AFM surface scans. Moreover, several methods for detailed analysis and comparison of surface topographies in simulated and experimental AFM images are provided. We demonstrate the applicability of the developed tools in the interpretation of high-speed AFM observations of proteins. The toolbox is implemented into the versatile interactive interface of the BioAFMviewer, which is a free software package available at www.bioafmviewer.com.

High-speed atomic force microscopy (HS-AFM) is a powerful tool for visualizing the dynamics of individual biomolecules. However, in single-molecule HS-AFM imaging applications, x,y-scanner ranges are typically restricted to a few hundred nanometers, preventing overview observation of larger molecular assemblies, such as 2-dimensional protein crystal growth or fibrillar aggregation. Previous advances in scanner design using mechanical amplification of the piezo-driven x,y-positioning system have extended the size of HS-AFM image frames to several tens of micrometer, but these large scanners may suffer from mechanical instabilities at high scan speeds and only record images with limited pixel numbers and comparatively low lateral resolutions (> 20–100 nm/pixel), complicating single-molecule analysis. Thus, AFM systems able to image large sample areas at high speeds and with nanometer resolution have still been missing. Here, we describe a HS-AFM sample-scanner system able to record large topographic images (≤ 36 × 36 µm2) containing up to 16 megapixels, providing molecular resolution throughout the image frame. Despite its large size, the flexure-based scanner features a high resonance frequency (> 2 kHz) and delivers stable operation even at high scans speeds of up to 7.2 mm/s, minimizing the time required for recording megapixel scans. We furthermore demonstrate that operating this high-speed scanner in time-lapse mode can simultaneously identify areas of spontaneous 2-dimensional Annexin A5 crystal growth, resolve the angular orientation of large crystalline domains, and even detect rare crystal lattice defects, all without changing scan frame size or resolution. Dynamic processes first identified from overview scans can then be further imaged at increased frame rates in reduced scan areas after switching to conventional HS-AFM scanning. The added ability to collect large-area, high-resolution images of complex samples within biological-relevant time frames extends the capabilities of HS-AFM from single-molecule imaging to the study of large dynamic molecular arrays. Moreover, large-area HS-AFM scanning can generate detailed structural data sets from a single scan, aiding the quantitative analysis of structurally heterogenous samples, including cellular surfaces.

Cyclic nucleotide-gated (CNG) channels are key to the signal transduction machinery of certain sensory modalities both in vertebrate and invertebrate organisms. They translate a chemical change in cyclic nucleotide concentration into an electrical signal that can spread through sensory cells. Despite CNG and voltage-gated potassium channels sharing a remarkable amino acid sequence homology and basic architectural plan, their functional properties are dramatically different. While voltage-gated potassium channels are highly selective and require membrane depolarization to open, CNG channels have low ion selectivity and are not very sensitive to voltage. In the last few years, many high-resolution structures of intact CNG channels have been released. This wealth of new structural information has provided enormous progress toward the understanding of the molecular mechanisms and driving forces underpinning CNG channel activation. In this review, we report on the current understanding and controversies surrounding the gating mechanism in CNG channels, as well as the deep intertwining existing between gating, the ion permeation process, and its modulation by membrane voltage. While the existence of this powerful coupling was recognized many decades ago, its direct structural demonstration, and ties to the CNG channel inherent pore flexibility, is a recent achievement.

Comment in Direct visualization of ion-channel gating in a native environment. [Proc Natl Acad Sci U S A. 2018]; International audience; Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated, cation-selective channel, is a prokaryotic homolog of the pentameric Cys-loop receptor ligand-gated ion channel family. Despite large changes in ion conductance, small conformational changes were detected in X-ray structures of detergent-solubilized GLIC at pH 4 (active/desensitized state) and pH 7 (closed state). Here, we used high-speed atomic force microscopy (HS-AFM) combined with a buffer exchange system to perform structural titration experiments to visualize GLIC gating at the single-molecule level under native conditions. Reference-free 2D classification revealed channels in multiple conformational states during pH gating. We find changes of protein-protein interactions so far elusive and conformational dynamics much larger than previously assumed. Asymmetric pentamers populate early stages of activation, which provides evidence for an intermediate preactivated state.

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., Cyclic nucleotide-gated (CNG) ion channels are non-selective cation channels key to signal transduction, but conformational changes associated with gating remained unknown. Here authors use high-speed atomic force microscopy to visualize SthK channels dynamics in response to cyclic nucleotides.

International audience; The 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.

Mechanical unfolding of proteins consisting of repeat domains is an excellent tool to obtain large statistics. Force spectroscopy experiments using atomic force microscopy 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 that have recently emerged minimize this uncertainty and would be excellent tools to probe unfolding history and rare events. However, detailed 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 provides 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 forces required to unfold two molecules pulled in parallel, difficult when using unspecific approaches. The proposed method represents a step forward toward more reproducible measurements to probe protein unfolding history and opens the door to systematic probing of rare and multiple molecule unfolding mechanisms.

The determination at atomic resolution of the three-dimensional molecular structure of membrane proteins such as receptors and several ion channels has been a major breakthrough in structural biology. The molecular structure of several members of the superfamily of voltage-gated ionic channels such as K+ and Na+ is now available. However, despite several attempts, the molecular structure at atomic resolution of the full cyclic nucleotide-gated (CNG) ion channel, although a member of the same superfamily of voltage-gated ion channels, has not been obtained yet, neither by X-ray crystallography nor by electron cryomicroscopy (cryo-EM). It is possible that CNG channels have a high structural heterogeneity, making difficult crystallization and single-particle analysis. To address this issue, we have combined single-molecule force spectroscopy (SMFS) and electrophysiological experiments to characterize the structural heterogeneity of CNGA1 channels expressed in Xenopus laevis oocytes. The unfolding of the cytoplasmic domain had force peaks, occurring with a probability from 0.2 to 0.96. Force peaks during the unfolding of the transmembrane domain had a probability close to 1, but the distribution of the increase in contour length between two successive force peaks had multiple maxima differing by tens of nanometers. Concomitant electrophysiological experiments showed that the rundown in mutant channels S399C is highly variable and that the effect of thiol reagents when specific residues were mutated was consistent with a dynamic structural heterogeneity. These results show that CNGA1 channels have a wide spectrum of native conformations that are difficult to detect with X-ray crystallography and cryo-EM.

Cyclic Nucleotide Gated (CNG) channels underlie sensory transduction in photoreceptors and olfactory epithelium.They belong to the family of voltage-gated ion channels, but open following binding of cyclic nucleotides (CNs).While electrophysiology combined with mutagenesis have identified the channel pore and the binding domain for CNs, conformational changes associated with gating have remained elusive. In this study we combine electrophysiology, site-directed mutagenesis, homology modelling and single molecule force spectroscopy (SMFS) to determine the molecular mechanisms underlying gating. Gating is mediated by electrostatic interactions between aspartate D413 in the C-linker and arginine R297 in the S4-S5 linker. Indeed when residues in position 413 and 297 are cross-linked, the mutant channels are locked in the open state. These electrostatic interactions induce an upward motion combined with a small rotation of the transmembrane α-helix S5 leading to conformational rearrangements of residues flanking the pore so that the lumen of the pore widens and the channel opens. Therefore gating of CNG channels is not mediated by a motion of the transmembrane α-helix S6 as in usual voltage gated Na+,K+ and Ca2+ channels, but by interactions between the cytoplasmic domain and the S4-S5 linker, possibly shared also by HCN channels (Decher et al., 2004).

Cyclic nucleotide-gated (CNG) channels and K+ channels have a significant sequence identity and are thought to share a similar 3D structure. K+ channels can accommodate simultaneously two or three permeating ions inside their pore and therefore are referred to as multi-ion channels. Also CNGA1 channels are multi-ion channels, as they exhibit an anomalous mole fraction effect (AMFE) in the presence of mixtures of 110 mm Li+ and Cs+ on the cytoplasmic side of the membrane. Several observations have identified the ring of Glu363 in the outer vestibule of the pore as one of the binding sites within the pore of CNGA1 channels. In the present work we identify a second binding site in the selectivity filter of CNGA1 channels controlling AMFE. Here, we show also that Cs+ ions at the intracellular side of the membrane block the entry of Na+ ions. This blockage is almost completely removed at high hyperpolarized voltages as expected if the Cs+ blocking site is located within the transmembrane electric field. Indeed, mutagenesis experiments show that the block is relieved when Thr359 and Thr360 at the intracellular entrance of the selectivity filter are replaced with an alanine. In T359A mutant channels AMFE in the presence of intracellular mixtures of Li+ and Cs+ is still present but is abolished in T360A mutant channels. These results suggest that the ring of Thr360 at the intracellular entrance of the selectivity filter forms another ion binding site in the CNGA1 channel. The two binding sites composed of the rings of Glu363 and Thr360 are not independent; in fact they mediate a powerful coupling between permeation and gating, a specific aspect of CNG channels.

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., Author summary Cyclic Nucleotide Gated (CNG) channels are nonselective cation channels with a key role in sensory transduction. Despite sharing a high homology with K+ channels, they allow the passage of large compounds like dimethylammonium (DMA) which are not permeable through K+ channels. We demonstrate that the conduction mechanism of this compound is radically different from the textbook scenario, in which an ion, in order to diffuse through the channel, must cross a series of barriers, whose height is possibly perturbed by the presence of other ions in the channel. We show that permeation of large cations in CNG is due to the destabilization of the pore induced by the simultaneous presence of two ions in the channel.

Significance Cyclic nucleotide-gated (CNG) channels underlie sensory transduction in photoreceptors and olfactory epithelium and share a high degree of homology with K + channels. However, these channels conduct Na + and K + differently: although K + channels discriminate with high accuracy Na + from K + , CNG channels do not discriminate among different cations. By combining electrophysiology, molecular dynamics simulations, and X-ray crystallography we found that the pore region exhibits a dynamic structure. We show that ( i ) the selectivity filter can adapt to large and small ions with a different geometry and ( ii ) the pore diameter critically depends on the ion within. We conclude that the pore of CNG channels is highly flexible and that this flexibility is at the basis of their poor ionic selectivity.

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.

Cyclic nucleotide-gated channels belong to the family of voltage-gated ion channels, but pore opening requires the presence of intracellular cyclic nucleotides. In the presence of a saturating agonist, cyclic nucleotide-gated channel gating is voltage independent and it is not known why cyclic nucleotide-gated channels are voltage-insensitive despite harbouring the S4-type voltage sensor. Here we report that, in the presence of Li(+), Na(+) and K(+), the gating of wild-type cyclic nucleotide-gated A1 and native cyclic nucleotide-gated channels is voltage independent, whereas their gating is highly voltage-dependent in the presence of Rb(+), Cs(+) and organic cations. Mutagenesis experiments show that voltage sensing occurs through a voltage sensor composed of charged/polar residues in the pore and of the S4-type voltage sensor. During evolution, cyclic nucleotide-gated channels lose their voltage-sensing ability when Na(+) or K(+) permeate so that the vertebrate photoreceptor cyclic nucleotide-gated channels are open at negative voltages, a necessary condition for phototransduction.

Gating of cyclic nucleotide gated (CNG) channels is thought not to be voltage dependent. However, in the presence of symmetrical Rb+ and Cs+ CNGA1 channels exhibit a clear voltage and time dependent gating of macroscopic current. In symmetrical Rb+ and Cs+ the single channel conductance (γsc) and the open probability (Pop) of w.t. CNGA1 channels are highly voltage dependent: Pop increases 2-4 times at positive voltages and γsc is larger at negative voltages. In the presence of Na+ and K+, rapid channel closures and the observed residual voltage dependence of γsc and the Pop are eliminated when the S4 domain is blocked by anchoring to it large sulphydryl reagents. γsc becomes very similar at positive and negative voltages when Glu363 in the pore is replaced by Alanine. These result uncover two major properties of CNGA1 channels, not yet properly evidentiated. Firstly, CNGA1 channels, in contrast to what usually believed, sense voltage and the S4 domain plays an important role in voltage sensing. Secondly, permeation and gating are coupled through a voltage sensor and that Glu363 is the major constituent of it.

We have analysed the permeation of divalent cations through homomeric CNGA1 and heteromeric CNGA1 and CNGB1 channels when 110 mM NaCl were in the patch pipette and 76.6 mM of CaCl2, MgCl2, SrCl2 and BaCl in the medium bathing the intracellular side of the patch. For homomeric CNGA1 channels the reversal potential Vrev was −6.5±2.5, −5.5±3.2, 21.3±7.2 and 48.6±8.5 mV and for heteromeric CNGA1 and CNGB1 channels Vrev was −1.5±0.9, 5.1±2.4, 7±3.2 and 2.3±1.2 mV for Ca2+, Mg2+, Sr2+ and Ba2+ respectively. For homomeric CNGA1 channels, the ratio of the current at +200 mV carried by Na+ and Ca2+, Mg2+, Sr2+ and Ba2+ was 0.05±0.03, 0.045±0.02,0.1±0.02 and 0.08±0.035 respectively. For heteromeric CNGA1 and CNGB1 these ratios were 0.18±0.04, 0.08±0.03, 0.21±0.05 and 0.13±0.03 respectively. At negative voltages divalent cations blocked the inward Na+ current more powerfully in homomeric than in heteromeric channels. These results show that divalent cations permeate more easily through heteromeric than homomeric CNG channels and that they block less powerfully the current carried by monovalent cations in hetoromeric channels.

In cyclic nucleotide-gated (CNGA1) channels, in the presence of symmetrical ionic conditions, current-voltage (I-V) relationship depends, in a complex way, on the radius of permeating ion. It has been suggested that both the pore and S4 helix contribute to the observed rectification. In the present manuscript, using tail and gating current measurements from homotetrameric CNGA1 channels expressed in Xenopus oocytes, we clarify and quantify the role of the pore and of the S4 helix. We show that in symmetrical Rb(+) and Cs(+) single-channel current rectification dominates macroscopic currents while voltage-dependent gating becomes larger in symmetrical ethylammonium and dimethylammonium, where the open probability strongly depends on voltage. Isochronal tail currents analysis in dimethylammonium shows that at least two voltage-dependent transitions underlie the observed rectification. Only the first voltage-dependent transition is sensible to mutation of charge residues in the S4 helix. Moreover, analysis of tail and gating currents indicates that the number of elementary charges per channel moving across the membrane is less than 2, when they are about 12 in K(+) channels. These results indicate the existence of distinct mechanisms underlying rectification in CNG channels. A restricted motion of the S4 helix together with an inefficient coupling to the channel gate render CNGA1 channels poorly sensitive to voltage in the presence of physiological Na(+) and K(+).

Voltage-gated Na+, K+, Ca2+ and cyclic nucleotide-gated (CNG) channels belong to the same super-family of ion channels. All these channels harbour a voltage sensor but their gating is different: the opening of CNG channels is poorly dependent on membrane voltage, whereas the gating of Na+, K+, Ca2+ channels is highly voltage-dependent. By using electrical recordings in voltage clamp mode, we show that in wild type CNGA1 channels the voltage sensor does not move as in K+ channels. However, when the hydrophobic interaction coupling Phe380 in S6 to Leu356 in the P-helix is impaired, the voltage sensor moves as in K+ channels and the gating of mutant channels F380A and L356A becomes voltage dependent. Voltage gating in mutant channels F380A is accompanied by nonlinear capacitative transients at the beginning and termination of voltage stimuli, reminiscent of gating currents measured in Kv channels, suggesting dipole rearrangement or charge translocation caused by the applied electrical field. Moreover, when Arg272 and Arg275 in the S4 segment are neutralized one at time in a F380A background, significant changes of the characteristic activation parameters can be observed, in agreement with what described for Kv and Nav channels. Therefore, we can conclude that the voltage sensor in CNG channels is blocked by the tight coupling between the P-helix and S6, necessary condition that makes the channel gated by binding of cyclic nucleotides and not by changes of membrane voltage.

CNG channels are permeable to alkali monovalent and divalent cations and to small organic cations. Therefore, CNG channels have a low ionic selectivity, attributed to an intrinsic flexibility of the filter region (Laio & Torre, 1999) mediating the coupling between permeation and gating (Gamel & Torre 2000; Holmgren, 2003; Kush, 2004). We have analysed in more detail the permeation of Cs+ in WT CNGA1 channels. In symmetrical Na+ or K+ conditions and in the presence of saturating cGMP concentrations the ratio between the current at +200 and −200 mV I200/I-200 is 1.3. In contrast, in the presence of symmetrical Cs+ I200/I-200 it is about 0.75. Under these conditions, single channel recordings reveal a surprising behaviour: the single channel conductance for Cs+ ions is about 18 pS at −180 mV, but becomes less than 5 pS at voltages larger than 100 mV. The open probability at −180 mV is about 0.2 and becomes close to 1 at +100 mV. When Thr360 is mutated to alanine, the single channel conductance for Cs+ ions is around 15 pS both at +100 and −100 mV. These results confirm the notion that the pore region of CNGA1 channels is highly flexible and that permeating ions modify and control channel gating. These results show also that the pore region of CNGA1 channels acts as a voltage sensor and modifies their conformation in response to changes of the applied field. The molecular mechanisms involved in these rearrangements are likely to consist in reorientation of electric dipoles, such as those of Thr360.

International audience; Single-molecule force spectroscopy (SMFS) measurements allow for quantification of the molecular forces required to unfold individual protein domains. Atomic force microscopy (AFM) is one of the long-established techniques for force spectroscopy (FS). Although FS at conventional AFM pulling rates provides valuable information on protein unfolding, in order to get a more complete picture of the mechanism, explore new regimes, and combine and compare experiments with simulations, we need higher pulling rates and μs-time resolution, now accessible via high-speed force spectroscopy (HS-FS). In this chapter, we provide a step-by-step protocol of HS-FS including sample preparation, measurements and analysis of the acquired data using HS-AFM with an illustrative example on unfolding of a well-studied concatamer made of eight repeats of the titin I91 domain.

Using an engineered cyclic nucleotide-gated (CNG) channel mimic based on the bacterial non-selective NaK channel, we examined how different ionic species interact with the pore by means of electrophysiology, all-atoms molecular dynamics simulations and X-ray crystallography.Difference and anomalous difference electron-density maps of high-resolution crystal structures (up to 1.85A) obtained after soaking with different ionic species (Li+, Na+, K+, Rb+, Cs+, Methylammonium) clearly shows the presence of different peaks inside the pore. Depending on these ionic species Glu66 and Thr67, located at the extracellular entrance of the selectivity filter, adopt different rotameric states. In the presence of Methylammonium, Glu66 points towards Tyr55 of the same subunit, while in the presence of Na+, Glu66 forms H-bonds with Thr67 and Thr60 of a neighboring subunit (Derebe et al, 2011, PNAS). Moreover, large-scale molecular dynamics simulations in a hydrated lipid bilayer at 0 mV indicates that Glu66 and Thr67 side chains are capable of large structural fluctuations which are modulated by the nature of the ion residing in the pore. In particular, in presence of Rb+ and Cs+ Glu66 explores at least three different conformations, characterized by a significantly different distribution of the local charge. Single channel recordings from this chimeric ionic channel in symmetrical Rb+ and Cs+ conditions show a strong voltage dependency similar to what observed in the native CNGA1 channel (Marchesi et al, 2012, Nat. Communications). These results indicate that membrane voltage catalyzes a conformational change of these residues, especially of Glu66, perturbing the hydrogen bonding network behind the selectivity filter. Non-selective ionic permeation emerges as a robust feature of an intrinsic flexible pore and is a key microscopic factor controlling ionic selectivity in CNG and possibly in other non-selective channels.

Cyclic nucleotide-gated (CNG) channels are members of the superfamily of voltage gated ion channels, but in the presence Na+ and K+ they are gated primarily by cyclic nucleotides (CNs) and only weakly by voltage. Here, we show that when extracellular pH (pHo) is decreased from pH 7.4 to 5 WT CNGA1 channels desensitize, i.e. the current activated by a steady cGMP concentration declines within 5-40 s by 20-80% in a voltage dependent way. Current desensitization is completely reversible upon removal of cGMP and voltage dependency of desensitization is associated to the displacement of 0.3 equivalent electronic charges across the electrical field. A very similar desensitization is observed in several mutant channels, such as E363A and T364A at the usual pH 7.4. At the desensitized state, the I/V relations are outwardly rectifying similarly in the WT CNGA1 channels at pH 5 and mutant channels E363A at pH 7.4. In the presence of symmetrical Rb+ or Cs+, the single channel conductance gsc in WT CNGA1 channels is highly voltage dependent and this voltage dependence is abolished in mutant channels E363A and T364A. CNGA1 channels have structurally-distinct types of voltage sensors, formed by charged and polar residues located in the pore, by the usual S4 voltage sensor and by an additional voltage sensor associated to the voltage dependency of desensitization. Our results not only shed a new light on voltage gating in CNG channels, but uncover the existence of a novel component of phototrasduction: neuronal signalling within the vertebrate retina is highly dependent on the pHo and proton elevation could act as a negative feedback.