Napolitano LMR et al. (2018), The permeation mechanism of organic cations thr...

XB-ART-55596

PLoS Comput Biol 2018 Aug 01;148:e1006295. doi: 10.1371/journal.pcbi.1006295.

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The permeation mechanism of organic cations through a CNG mimic channel.

Napolitano LMR , Marchesi A , Rodriguez A , De March M , Onesti S , Laio A , Torre V .

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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.

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Species referenced: Xenopus laevis
Genes referenced: cnga1 mhc2-dma

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	Fig 1. Crystal structure of the CNG-mimic:DMA complex.Overview of the AABB and CCDD NaK2CNG tetrameric structures (PDB ID: 4R7C) showing only two monomers for clarity; residues in the selectivity filter are shown as sticks. The black box indicates the region expanded in the right panel, corresponding to the selectivity filter. On the right of each molecule, the one-dimensional electron density profile along the axis of symmetry is reported. The DMA cation is shown in cyan and well-ordered water molecules are shown as red spheres. The crystallographic sites are labeled S0-4c, starting from the extracellular side. The final refined 2Fo-Fc electron density maps are shown at 1.3Ïƒ in light blue mesh. The position of Val64 is indicated.
	Fig 2. DMA permeation pathway.(A) Free Energy profile (kJ/mol) along the distance of the DMA from its binding site, represented by center of mass of Val64 (nm). The different colors indicate the runs in which the whole BE-META has been divided (See Materials and methods). The different free energy minima are indicated as S4MD, S3MD, SXMD and S0MD. All the free energy profiles are shown with the relative error bars estimated by block analysis [29] (B) Position and interaction of the DMA ions in the different minima along the permeation pathway in the CNG-mimic selectivity filter, as modelled by MD. DMA is depicted in stick, while only the CÎ± trace of the selectivity filter is shown. (C) Close-up of the MD structures corresponding to the minima shown in (A-B). The residues forming hydrogen bonds with DMA (Thr63; Val64; Gly65; Glu66 and Tyr55) and the DMA itself have been drawn as sticks. The black dashed lines refer to H-bonds, while the red circles indicate the hydrophobic interactions. (D) Electron density map for the selectivity filter for of the AABB and CCDD tetramers (as in Fig 1) where the electron density peaks previously assigned to water molecules have been replaced by weakly bound DMA cations, based on the MD results.
	Fig 3. The presence of a second DMA destabilizes the first one.(A) and (B) Left side: Representative structures of the MD simulations performed in the presence of a single DMA in S3 (A) and two DMA (B). For simplicity only the filter domain (Thr62-Pro68) of the NaK2CNG channel is shown. DMA has been drawn as cyan sticks while the amino acids residues as yellow sticks. (A) and (B) Right side: Free Energy profile (kJ/mol) along the distance of the DMA from its binding site, represented by center of mass of Val64 (nm), in the case of a single DMA (A) and in the DMA-DMA system (B). (C) Position and interaction of the DMA ions in the global minimum along the permeation pathway in the CNG-mimic selectivity filter, as modelled by MD. DMA is depicted in stick, while only the CÎ± trace of the selectivity filter is shown. In i) and ii) panels, close-up of the MD structures corresponding to the DMA-DMA global minimum. The residues and the DMA itself have been drawn as sticks. The residues forming hydrogen bonds with DMA are indicated (Thr63 and Gly65). The black dashed lines refer to H-bonds.
	Fig 4. Currents activated by 1 mM cGMP in the presence of different voltage commands and amounts of DMA.(A) Representative currents observed in the presence of different amounts (indicated in the figure) of intracellular DMA (DMAi) at +200 mV (holding potential of 0 mV). The dashed line indicates the zero-current level. Currents are normalized to the mean current observed in the presence of 110 mM DMAi and the solution filling the patch pipette contained 110 mM DMA. (B) Dependence of the cGMP-gated conductance on the ionic activity of DMAi at +140 (filled triangles), +160 (open squares), +180 (filled circles) and +200 mV (open triangle). Conductances are normalized to the conductance observed at +200 mV in the presence of 110 mM DMAi. Each point is the average obtained from at least three patches. These data have been fitted with the Eq (3) obtaining an apparent Kd = 52 mM. Data are presented as mean +/- SD (n = 4).
	Fig 5. Model for the DMA permeation through the CNGA1 channel.The first scheme (I site) describes a simple model of DMA permeation in which there is a single binding site, while the second scheme (II sites) depicts a different scenario with the CNGA1 channel occupied simultaneously by two ions referred to as right (R) and left (L). PE is the probability that the channel is empty, PO refers to the channel occupied; PL is the probability that the channel is occupied in the left site, PR is occupied in the right side, PD when the channel is occupied by two ions. kL is the rate for the transition in which the ion enters into the channel, while kR for the transition in which the ion leaves the channel crossing the barrier towards the right. The rate kR~ is associated to a transition between the state with the Double Occupancy and the state with a single ion in the Left side. k+ is the transition rate from the binding site at the left to that at the right and k- is the corresponding reverse rate. The transition rates kR, kL, k+ and k- are estimated from the Free Energy profile computed with a single DMA inside the pore (see right panel in Fig 3A) and the transition rate kR~ is estimated from the Free Energy profile computed with a second DMA inside the pore (see right panel in Fig 3B).

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