Mechanism of Ionic Permeation in the Mimics of CNG Channels: A Structural, Functional and Computational Analysis

Abstract

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.