Arin Marchesi

Gating in CNGA1 channels

The aminoacid sequences of CNG and K+ channels share a significant sequence identity, and it has been suggested that these channels have a common ancestral 3D architecture. However, K+ and CNG channels have profoundly different physiological properties: indeed, K+ channels have a high ionic selectivity, their gating strongly depends on membrane voltage and when opened by a steady depolarizing voltage several K+ channels inactivate, whereas CNG channels have a low ion selectivity, their gating is poorly voltage dependent, and they do not desensitize in the presence of a steady concentration of cyclic nucleotides that cause their opening. The purpose of the present review is to summarize and recapitulate functional and structural differences between K+ and CNG channels with the aim to understand the gating mechanisms of CNG channels.

A structural, functional, and computational analysis suggests pore flexibility as the base for the poor selectivity of CNG channels.

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. Cyclic nucleotide-gated (CNG) ion channels, despite a significant homology with the highly selective K+ channels, do not discriminate among monovalent alkali cations and are permeable also to several organic cations. We combined electrophysiology, molecular dynamics (MD) simulations, and X-ray crystallography to demonstrate that the pore of CNG channels is highly flexible. When a CNG mimic is crystallized in the presence of a variety of monovalent cations, including Na+, Cs+, and dimethylammonium (DMA+), the side chain of Glu66 in the selectivity filter shows multiple conformations and the diameter of the pore changes significantly. MD simulations indicate that Glu66 and the prolines in the outer vestibule undergo large fluctuations, which are modulated by the ionic species and the voltage. This flexibility underlies the coupling between gating and permeation and the poor ionic selectivity of CNG channels.

Gating of cyclic nucleotide-gated channels is voltage dependent

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.

Multiple mechanisms underlying rectification in retinal cyclic nucleotide‐gated (CNGA1) 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+.

The gating mechanism in cyclic nucleotide-gated ion channels

Cyclic nucleotide-gated (CNG) channels mediate transduction in several sensory neurons. These channels use the free energy of CNs’ binding to open the pore, a process referred to as gating. CNG channels belong to the superfamily of voltage-gated channels, where the motion of the α-helix S6 controls gating in most of its members. To date, only the open, cGMP-bound, structure of a CNG channel has been determined at atomic resolution, which is inadequate to determine the molecular events underlying gating. By using electrophysiology, site-directed mutagenesis, chemical modification, and Single Molecule Force Spectroscopy, we demonstrate that opening of CNGA1 channels is initiated by the formation of salt bridges between residues in the C-linker and S5 helix. These events trigger conformational changes of the α-helix S5, transmitted to the P-helix and leading to channel opening. Therefore, the superfamily of voltage-gated channels shares a similar molecular architecture but has evolved divergent gating mechanisms.

A ring of threonines in the inner vestibule of the pore of CNGA1 channels constitutes a binding site for permeating ions

•  Cyclic nucleotide‐gated (CNG) channels are multi‐ion channels showing the anomalous mole fraction effect (AMFE) in the presence of Li+ and Cs+ mixtures. •  We show that Cs+ ions at the intracellular side of the membrane block the entry of Na+ ions in a voltage dependent way. •  The blockage is relieved when Thr359 and Thr360 at the intracellular entrance of the selectivity filter are replaced with an alanine. Moreover, the AMFE in the presence of intracellular mixtures of Li+ and Cs+ is abolished in T360A mutant channels. •  We have identified a second binding site – composed by the ring of Thr360 at the intracellular vestibule – in the selectivity filter of CNG channels controlling monovalent cations selectivity and permeation. •  These results help us understand fundamental similarities and differences between the pore of CNG channels and K+ channels.

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

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.

Structural titration of receptor ion channel GLIC gating by HS-AFM

Significance Gloeobacter violaceus ligand-gated ion channel (GLIC) is a proton-gated, cation-selective channel, a Cys-loop family member of the pentameric ligand-gated ion channels (pLGICs). In a pursuit to provide evidence of function-related conformational changes of a pLGIC in membrane, at ambient temperature and pressure and of the very same unlabeled molecules, we used high-speed atomic force microscopy (HS-AFM) combined with a buffer exchange system to visualize reversible changes of the supramolecular arrangement and conformations of GLIC during pH changes: a structural titration experiment. Reference-free classification of the molecules assigns conformations to states during the pH titration. We find large conformational changes with an unexpected closed-state structure with tightened extracellular domains. Furthermore, we provide evidence for the short-term existence of asymmetric channels at early stages of activation. 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.

Structural Heterogeneity of CNGA1 Channels Revealed by Electrophysiology and Single-Molecule Force Spectroscopy

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.

Proton transfer unlocks inactivation in cyclic nucleotide‐gated A1 channels

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.