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 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.
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.CNG channels | pore flexibility | X-ray crystallography | MD simulations I n K + selective channels, the opening and closing of the ion channel pore (gating) and the translocation of ions through the pore (permeation) are considered independent processes with distinct structural basis (1). Gating is controlled by the bundle crossing at the intracellular side, whereas permeation reflects ion-ion and ion-pore interactions within the selectivity filter (1-5). Based on these experimental observations, the current paradigm assumes that the 3D structure of the selectivity filter is relatively rigid during ion translocation and the mechanisms of ionic permeation can be deduced in essence from its crystal structure. This paradigm has been successfully applied to several K + channels (4, 6, 7). Cyclic nucleotide-gated (CNG) channels underlie sensory transduction in the retina and olfactory epithelium and share a high degree of homology with K + channels (8-10). In contrast to K + channels, CNG channels' primary gate is located at the selectivity filter (11), suggesting that the same protein region controls ion permeation and gating. In CNG channels the ionic species present inside the pore influences channel gating; however, the nature of this coupling is not well understood (12-16). In the presence of large cations, such as Rb + and Cs + , channel conductance and gating are also controlled by membrane voltage, and current-voltage relationships activated by 1 mM cGMP depend on the radius of the permeating ion (17, 18) (Fig. S1).Structural information on CNG channels is limited to a lowresolution electron microscopy map (19), partial crystal structures of the intracellular cyclic nucleotide-binding domains (20)(21)(22), and the crystal structure of a chimeric channel in which the CNG selectivity-filter sequence is engineered into a bacterial NaK channel, creating a CNG mimic (NaK2CNG; Fig. 1 A and B) that shares several properties of CNG channels (23). This CNG mimic provides a suitable model for understanding the properties of the pore underlying the low ionic selectivity and the coupling between gating and perme...
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.
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