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

Abstract

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...

Voltage vs. Ligand I: Structural basis of the intrinsic flexibility of S3 segment and its significance in ion channel activation

“…On the other hand, also in CNG channels, some detectable V-dependence has been described, particularly in the presence of large permeant cations [65]. Importantly, this peculiarity has been associated with the intrinsic pore flexibility reported in those channels [66]. In view of these findings, we decided to further explore if S3 flexibility is in some way correlated with V-dependence as a function of additional activators including diverse chemical compounds or "ligands", the mechanical force exerted along the membrane, or the effect of temperature.…”

Structure of a eukaryotic cyclic-nucleotide-gated channel

“…The close proximity of E379 side chains is consistent with a previous finding in a vertebrate CNG channel that a single protonation site is made by two glutamates 31 . On the other hand, the luminal projection of the E379 side chain is in contrast with the side chain orientation of the analogous glutamate in NaK2CNG-E, an engineered chimeric NaK channel containing four amino acids (ETPP) of the CNG channel selectivity filter sequence 32–34 (Extended Data Fig. 9c).…”

“…9c). In NaK2CNG-E, the glutamate side chain points toward the protein interior and is engaged in dynamic protein packing around the selectivity filter 32–34 .…”

The gating mechanism in cyclic nucleotide-gated ion channels

“…This hypothesis, however, was not confirmed by other experiments, which located the gate in the selectivity filter 10 , 11 . Thus, the same region of CNGA1 channels controls ion permeation and gating, leading to a more complex gating mechanism 12 – 15 .…”