NaK Channel Research Articles

Mechanism of Calcium Permeation in a Glutamate Receptor Ion Channel.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are neurotransmitter-activated cation channels ubiquitously expressed in vertebrate brains. The regulation of calcium flux through the channel pore by RNA-editing is linked to synaptic plasticity while excessive calcium influx poses a risk for neurodegeneration. Unfortunately, the molecular mechanisms underlying this key process are mostly unknown. Here, we investigated calcium conduction in calcium-permeable AMPAR using Molecular Dynamics (MD) simulations with recently introduced multisite force-field parameters for Ca2+. Our calculations are consistent with experiment and explain the distinct calcium permeability in different RNA-edited forms of GluA2. For one of the identified metal binding sites, multiscale Quantum Mechanics/Molecular Mechanics (QM/MM) simulations further validated the results from MD and revealed small but reproducible charge transfer between the metal ion and its first solvation shell. In addition, the ion occupancy derived from MD simulations independently reproduced the Ca2+ binding profile in an X-ray structure of an NaK channel mimicking the AMPAR selectivity filter. This integrated study comprising X-ray crystallography, multisite MD, and multiscale QM/MM simulations provides unprecedented insights into Ca2+ permeation mechanisms in AMPARs, and paves the way for studying other biological processes in which Ca2+ plays a pivotal role.

The potassium-selective channel "NaK2K": Why is it a potassium channel?

Potassium channels are transmembrane proteins that enable the flux of potassium ions across the lipid membranes. They are characterized by high conductance rates near the diffusion limit with K+ selectivity more than a thousandfold over Na+. The potassium-selective NaK channel mutant “NaK2K” was found permeable for Rb+ and Cs+ at poor rates despite the sub-angstrom difference in the atomic radius between those ions and K+. Although experimental studies have characterized the conductance and binding profiles of different monovalent ions to NaK2K, the reasons behind this remarkable K+ selectivity is not clear yet at the atomistic level. Aiming to delineate the mechanistic of K+ selectively in NaK2K, we performed a microsecond scale computational simulations of Rb+ and K+ permeations through NaK2K using two different forcefields. Consistent with the previous experimental studies, our simulations showed very poor permiation rates of Rb+ compared to K+ regardless of the force field used. Both of the ions were found to have different binding profiles to the NaK2K selectivity filter. To further investigate the reasons behind the low permeation rates of Rb+, another two sets of simulations were carried out after modifying the LJ potentials parameters of Rb+ to those of K+. Modifying both of the LJ parameters independently did not impact the Rb+ permeation rates. On the other hand, modifying both of them at the same time resulted in permeation rates of Rb+ similar to those of K+. These observations suggest that the ion mass has no effect on its permeation rate but that it is dominated by precise coordination geometry.

Conformational plasticity of NaK2K and TREK2 potassium channel selectivity filters

The K+ channel selectivity filter (SF) is defined by TxGYG amino acid sequences that generate four identical K+ binding sites (S1-S4). Only two sites (S3, S4) are present in the non-selective bacterial NaK channel, but a four-site K+-selective SF is obtained by mutating the wild-type TVGDGN SF sequence to a canonical K+ channel TVGYGD sequence (NaK2K mutant). Using single molecule FRET (smFRET), we show that the SF of NaK2K, but not of non-selective NaK, is ion-dependent, with the constricted SF configuration stabilized in high K+ conditions. Patch-clamp electrophysiology and non-canonical fluorescent amino acid incorporation show that NaK2K selectivity is reduced by crosslinking to limit SF conformational movement. Finally, the eukaryotic K+ channel TREK2 SF exhibits essentially identical smFRET-reported ion-dependent conformations as in prokaryotic K+ channels. Our results establish the generality of K+-induced SF conformational stability across the K+ channel superfamily, and introduce an approach to study manipulation of channel selectivity.

Full opening of helix bundle crossing does not lead to NaK channel activation.

A critical part of ion channel function is the ability to open and close in response to stimuli and thus conduct ions in a regulated fashion. While x-ray diffraction studies of ion channels suggested a general steric gating mechanism located at the helix bundle crossing (HBC), recent functional studies on several channels indicate that the helix bundle crossing is wide-open even in functionally nonconductive channels. Two NaK channel variants were crystallized in very different open and closed conformations, which served as important models of the HBC gating hypothesis. However, neither of these NaK variants is conductive in liposomes unless phenylalanine 92 is mutated to alanine (F92A). Here, we use NMR to probe distances at near-atomic resolution of the two NaK variants in lipid bicelles. We demonstrate that in contrast to the crystal structures, both NaK variants are in a fully open conformation, akin to Ca2+-bound MthK channel structure where the HBC is widely open. While we were not able to determine what a conductive NaK structure is like, our further inquiry into the gating mechanism suggests that the selectivity filter and pore helix are coupled to the M2 helix below and undergo changes in the structure when F92 is mutated. Overall, our data show that NaK exhibits coupling between the selectivity filter and HBC, similar to K+ channels, and has a more complex gating mechanism than previously thought, where the full opening of HBC does not lead to channel activation.

Interrogating the gating motions of the NaK channel.

Previous crystallographic studies depicted a physical gate of the NaK channel localized at a bundle crossing of pore-lining helices, but solution NMR studies in the current issue of JGP suggest otherwise.

Mechanistic Insights into Structural Stability of the Selectivity Filters in Typical Cation Channels

The reliable functioning of ion channels should be closely related to their structural stability. The selectivity filter in the KcsA potassium channel possesses four stable ion binding sites that can coordinate nearly fully dehydrated ions, whereas only two of such binding sites exist in the non-selective NaK channel, and none of them is found in the NavAb sodium channel. Here we show that the stability of the selectivity filters in these tetrameric cation channels is inversely correlated with the number of stable binding sites by extensive molecular dynamics simulations. While the presence of coordinated ions is crucial for the selectivity filters of the KcsA and NaK channels to stabilize the conformations in their crystal structures, the selectivity filter of the NavAb channel shows higher stability, independent of the presence of ions. We further show that the distinct repulsive electrostatic interactions between negatively charged oxygen atoms in the selectivity filter which form the stable binding sites are responsible for the different stability of these cation channels. The hydrogen bonding networks between residues in the selectivity filter and its adjacent pore helix also play an important role in maintaining stability. Together, these results provide important mechanistic insights into the structural stability of the selectivity filters in typical cation channels.

Sodium Ions Do Not Stabilize the Selectivity Filter of a Potassium Channel

Ion conduction is an essential function for electrical activity in all organisms. The non-selective ion channel NaK was previously shown to adopt two stable conformations of the selectivity filter. Here, we present solid-state NMR measurements of NaK demonstrating a population shift between these conformations induced by changing the ions in the sample while the overall structure of NaK is not affected. We show that two K+-selective mutants (NaK2K and NaK2K-Y66F) suffer a complete loss of selectivity filter stability under Na+ conditions, but do not collapse into a defined structure. Widespread chemical shift perturbations are seen between the Na+ and K+ states of the K+-selective mutants in the region of the pore helix indicating structural changes. We conclude that the stronger link between the selectivity filter and the pore helix in the K+-selective mutants, compared to the non-selective wild-type NaK channel, reduces the ion-dependent conformational flexibility of the selectivity filter.

Measurement of Selectivity Filter Dynamics in Selective and Non-Selective NaK Channel Variants

K+ channel selectivity is governed by the selectivity filter (SF), a region of the pore lined by backbone carbonyl moieties that contacts permeating ions. There is ongoing debate as to the relative importance of various SF features in producing K+-selectivity, such as ligand type, number of ion binding sites, structural organization of the binding sites, and rigidity of the SF structure. NaK, a non-selective ion channel from Bacillus cereus bears two of the four canonical K+ binding sites and can be made K+-selective by mutating the SF sequence to the canonical K+ SF sequence. This mutant, NaK2K, has an SF structure virtually identical to that of bona fide K+ channels, inspiring the hypothesis that four contiguous ion binding sites are necessary to confer K+ selectivity. However, when Y55 is mutated to F in NaK2K (NaK2K-Y55F), the SF structure remains the same as seen by X-ray crystallography, but the channel is not selective for K+ over Na+. The authors speculate that the NaK2K-Y55F SF is more dynamic in solution than the NaK2K SF, resulting in reduced binding site number and loss of K+ selectivity. This hypothesis can be tested by performing NMR spin relaxation measurements that are sensitive to ps-ns timescale dynamics. Data collected for NaK suggests that dynamics vary across the SF; the extracellular mouth is more dynamic than the intracellular base where the two conserved binding sites are located. It is expected that the dynamics of NaK2K-Y55F will mirror those of NaK, rather than NaK2K.

Selectivity filter modalities and rapid inactivation of the hERG1 channel

The human ether-á-go-go-related gene (hERG1) channel conducts small outward K+ currents that are critical for cardiomyocyte membrane repolarization. The gain-of-function mutation N629D at the outer mouth of the selectivity filter (SF) disrupts inactivation and K+-selective transport in hERG1, leading to arrhythmogenic phenotypes associated with long-QT syndrome. Here, we combined computational electrophysiology with Markov state model analysis to investigate how SF-level gating modalities control selective cation transport in wild-type (WT) and mutant (N629D) hERG1 variants. Starting from the recently reported cryogenic electron microscopy (cryo-EM) open-state channel structure, multiple microseconds-long molecular-dynamics (MD) trajectories were generated using different cation configurations at the filter, voltages, electrolyte concentrations, and force-field parameters. Most of the K+ permeation events observed in hERG1-WT simulations occurred at microsecond timescales, influenced by the spontaneous dehydration/rehydration dynamics at the filter. The SF region displayed conductive, constricted, occluded, and dilated states, in qualitative agreement with the well-documented flickering conductance of hERG1. In line with mutagenesis studies, these gating modalities resulted from dynamic interaction networks involving residues from the SF, outer-mouth vestibule, P-helices, and S5-P segments. We found that N629D mutation significantly stabilizes the SF in a state that is permeable to both K+ and Na+, which is reminiscent of the SF in the nonselective bacterial NaK channel. Increasing the external K+ concentration induced "WT-like" SF dynamics in N629D, in qualitative agreement with the recovery of flickering currents in experiments. Overall, our findings provide an understanding of the molecular mechanisms controlling selective transport in K+ channels with a nonconventional SF sequence.

Structural Studies of Mutants of the NaK Channel

Structural Studies of Mutants of the NaK Channel

A single NaK channel conformation is not enough for non-selective ion conduction

NaK and other non-selective channels are able to conduct both sodium (Na+) and potassium (K+) with equally high efficiency. In contrast to previous crystallographic results, we show that the selectivity filter (SF) of NaK in native-like lipid membranes adopts two distinct conformations that are stabilized by either Na+ or K+ ions. The atomic differences of these conformations are resolved by solid-state NMR (ssNMR) spectroscopy and molecular dynamics (MD) simulations. Besides the canonical K+ permeation pathway, we identify a side entry ion-conduction pathway for Na+ permeation unique to NaK. Moreover, under otherwise identical conditions ssNMR spectra of the K+ selective NaK mutant (NaK2K) reveal only a single conformational state. Therefore, we propose that structural plasticity within the SF and the selection of these conformations by different ions are key molecular determinants for highly efficient conduction of different ions in non-selective cation channels.

All atom NMDA receptor transmembrane domain model development and simulations in lipid bilayers and water.

N-methyl-d-aspartate receptors (NMDARs) are members of the ionotropic glutamate receptor family that mediate excitatory synaptic transmission in the central nervous system. The channels of NMDARs are permeable to Ca2+ but blocked by Mg2+, distinctive properties that underlie essential brain processes such as induction of synaptic plasticity. However, due to limited structural information about the NMDAR transmembrane ion channel forming domain, the mechanism of divalent cation permeation and block is understood poorly. In this paper we developed an atomistic model of the transmembrane domain (TMD) of NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors). The model was generated using (a) a homology model based on the structure of the NaK channel and a partially resolved structure of an AMPA receptor (AMPAR), and (b) a partially resolved X-ray structure of GluN1/2B NMDARs. Refinement and extensive Molecular Dynamics (MD) simulations of the NMDAR TMD model were performed in explicit lipid bilayer membrane and water. Targeted MD with simulated annealing was introduced to promote structure refinement. Putative positions of the Mg2+ and Ca2+ ions in the ion channel divalent cation binding site are proposed. Differences in the structural and dynamic behavior of the channel protein in the presence of Mg2+ or Ca2+ are analyzed. NMDAR protein conformational flexibility was similar with no ion bound to the divalent cation binding site and with Ca2+ bound, whereas Mg2+ binding reduced protein fluctuations. While bound at the binding site both ions retained their preferred ligand coordination numbers: 6 for Mg2+, and 7–8 for Ca2+. Four asparagine side chain oxygens, a back-bone oxygen, and a water molecule participated in binding a Mg2+ ion. The Ca2+ ion first coordination shell ligands typically included four to five side-chain oxygen atoms of the binding site asparagine residues, two water molecules and zero to two backbone oxygens of the GluN2B subunits. These results demonstrate the importance of high-resolution channel structures for elucidation of mechanisms of NMDAR permeation and block.

Dynamic Coupling between the Gates in the NaK Channel

Dynamic Coupling between the Gates in the NaK Channel

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.

Selectivity of a Singly Permeating Ion in Nonselective NaK Channel: Combined QM and MD Based Investigations

Ion channels, such as potassium channels are known to discriminate ions to achieve remarkable selective transportation of K(+) over Na(+) through the membrane. The recently reported NaK ion channel, on the contrary, seems to be an exception, as it is observed to permeate most of the group IA alkali metal cations and hence is suggested to be nonselective in nature. However, does that correspond to a complete annihilation of selectivity inside the selectivity filter (SF) of the channel? What is the origin of such nonselectivity/selectivity, if any? The present computational study is an extensive multiscale modeling approach to find the probable answers to these intriguing questions. Here, we have used density functional theory (DFT) based calculations using a realistic truncated model of SF from the crystal structures of the NaK ion channel to evaluate the binding of various alkali metal ions (Na(+), K(+) and Cs(+)), free from "contamination" due to the absence any other "rivalry" cations, in its different binding sites. Among all of the possible binding sites, a vestibule is noticed to be nonselective and seen to act as a probable binding site only in the presence of multiple ions. Binding sites S3 and S4 are found to be selective for K(+) and Na(+), respectively. As an important observation, we find that calculations on oversimplified models using an isolated ion binding site may lead to an erroneous selectivity trend as it neglects the synergetics of consecutive binding sites on the final outcome. Energy decomposition analysis revealed ion-dipole electrostatics as the major contributing interaction in metal-bound binding sites. Our investigations find that although NaK is permeable to monovalent alkali metal ions, strongly "site specific" selectivity does exist at the three well-defined noncontiguous binding sites of the SF. Different important physicomechanical parameters (such as ligating environment, synergistic influence of binding sites, and topological constraints) are found to be the determining factor to induce the "site specific" selectivity of ions during translocation. Wherever possible, our computed results are compared with the available experimental findings. We finally conduct a detailed umbrella sampling-corrected metadynamics simulation in order to obtain an ion permeation free energy landscape within the SF that corroborates well with the "site specific" selectivity trend.

Solution NMR to Investigate Gating in the NaK Channel

The NaK channel is small bacterial non-selective cation channel that has proved to be an excellent model system to study basic biochemical and biophysical question concerning ion channels. NaK has been crystalized in the closed state and as an open channel using a truncated construct (NaKΔ19) missing the M0 helix. NaK shares a homologous architecture to tradition K+ channel pores such as KcsA, but it is non selective. However, small mutation in the selectivity filter can change NaK into a K+ selective channel. Solution NMR spectra of NaK solubilized in isotropic bicelles show superior spectral resolution, allowing for detailed solution NMR studies of NaK structure and dynamics in bicelles. Regulation of NaK is currently unknown, however the location of the amphipathic M0 helix on the membrane surface packed against two adjacent monomers makes it a likely contributor to channel gating. Preliminary work suggests that the full length NaK channel exists in at least two states in slow exchange at 45oC. Mutations to the M0 helix shift the equilibrium to a single population, supporting a role for the M0 helix in determining the structural and functional state of the channel. NaKΔ19 NMR spectra are also sensitive to addition of amino acids just before the M1 helix. This suggests that this region connecting the M0 helix to the M1 helix is important in determining NaK dynamics. Comparison of our results with functional studies highlights specific interactions between the M0 helix and the pore domain that determine the structural and functional state of the channel.

K(+) preference at the NaK channel entrance revealed by fluorescence lifetime and anisotropy analysis of site-specifically incorporated (7-hydroxycoumarin-4-yl)ethylglycine.

The fluorescent unnatural amino acid, (7-hydroxycoumarin-4-yl)ethylglycine (HC), was site-specifically incorporated at the Phe69 site, close to the entrance of the selectivity filter of the NaK channel. Decreased fluorescence lifetime and elevated time-resolved anisotropy of NaK-F69HC in buffers with high K(+)/Na(+) molar ratios indicated the K(+) preference at the entrance of the NaK channel, consistent with previous crystal structure results of the NaK channel.

NMDA Receptor Transmembrane Domain: Structure and Mechanism of Ion Selectivity

NMDA receptors (NMDARs) are iGluR subfamilies that are activated during synaptic transmission. The voltage dependence of NMDARs differentiates them from other iGluRs: at typical neuronal resting voltages, NMDAR channels are mostly blocked by Mg2+, but when membrane voltage is depolarized, Mg2+ block is relieved, resulting in Ca2+ influx through NMDAR at postsynaptic sites. The mechanisms, by which NMDARs select Ca2+ for permeation over all other physiological ions, while binding Mg2+ and restricting its permeation, are not well understood.Recently, partially resolved medium-resolution structures of an NMDAR tetramer were published. The structures tremendously improve our knowledge and understanding of the architecture and design of the NMDARs. Yet, one of the key structural features, namely the ion selectivity filter and parts of the pore region of the ion channel itself, are missing from these structures. This region of the protein has not been resolved also in the earlier high-resolution structure of an AMPAR, a closely related iGluR family member.Previously a successful homology model of the NMDAR TMD using one of the potassium channel family members, NaK channel as a template, was shown to have good agreements with available experimental data [Siegler et. al., Nat Neurosci 15: 406–13]. Given recently released NMDAR structures and our previously developed homology model, we are now in position to propose a high resolution NMDAR TMD model that is based on a hybrid structure. We performed extensive molecular dynamics (MD) and targeted MD simulations of our NMDAR TMD domain model in lipid bilayer and water. We demonstrate that our proposed structure is stable in simulations and has a well-formed binding site for Mg2+ and Ca2+.

The conserved potassium channel filter can have distinct ion binding profiles: structural analysis of rubidium, cesium, and barium binding in NaK2K.

Potassium channels are highly selective for K(+) over the smaller Na(+). Intriguingly, they are permeable to larger monovalent cations such as Rb(+) and Cs(+) but are specifically blocked by the similarly sized Ba(2+). In this study, we used structural analysis to determine the binding profiles for these permeant and blocking ions in the selectivity filter of the potassium-selective NaK channel mutant NaK2K and also performed permeation experiments using single-channel recordings. Our data revealed that some ion binding properties of NaK2K are distinct from those of the canonical K(+) channels KcsA and MthK. Rb(+) bound at sites 1, 3, and 4 in NaK2K, as it does in KcsA. Cs(+), however, bound predominantly at sites 1 and 3 in NaK2K, whereas it binds at sites 1, 3, and 4 in KcsA. Moreover, Ba(2+) binding in NaK2K was distinct from that which has been observed in KcsA and MthK, even though all of these channels show similar Ba(2+) block. In the presence of K(+), Ba(2+) bound to the NaK2K channel at site 3 in conjunction with a K(+) at site 1; this led to a prolonged block of the channel (the external K(+)-dependent Ba(2+) lock-in state). In the absence of K(+), however, Ba(2+) acts as a permeating blocker. We found that, under these conditions, Ba(2+) bound at sites 1 or 0 as well as site 3, allowing it to enter the filter from the intracellular side and exit from the extracellular side. The difference in the Ba(2+) binding profile in the presence and absence of K(+) thus provides a structural explanation for the short and prolonged Ba(2+) block observed in NaK2K.

Molecular Strategies to Achieve Selective Conductance in NaK Channel Variants

A recent crystallization of several ion channels has provided strong impetus for efforts aimed at understanding the different strategies employed by nature for selective ion transport. In this work, we used two variants of the selectivity filter of NaK channel to explore molecular mechanisms that give rise to K(+)-selectivity. We computed one-dimensional (1D) and two-dimensional (2D) potentials of mean force (PMFs) for ion permeation across the channel. The results indicate that the energies for Na(+) and K(+) permeation across the selectivity filter display significant differences in positions of the binding sites and barriers. One characteristic signature of a K(+)-selective channel is the apparent preservation of the site analogous to that of S2 in KcsA. The S2-bound ion can be almost ideally dehydrated and coordinated by 6 to 8 carbonyls. In a striking contrast, the PMFs controlling transport of ions in a nonselective variant show almost identical profiles for either K(+) or Na(+) and significant involvement of water molecules in ion coordination across the entire selectivity filter. An analysis of differences in 1D PMFs for Na(+) and K(+) suggests that coordination number alone is an insufficient predictor of site selectivity, while chemical composition (ratio of carbonyls and water molecules) correlates well with preference for K(+). Multi-ion effects such as dependence of the barriers and wells for permeant ion on the type of copermeant ion were found to play a significant role in the selectivity signature of the channel as well.