Exploring the origin of the ion selectivity of the KcsA potassium channel

The availability of structural information about biological ion channels provides an opportunity to gain a detailed understanding of the control of ion selectivity by biological systems. However, accomplishing this task by computer simulation approaches is very challenging. First, although the activation barriers for ion transport can be evaluated by microscopic simulations, it is hard to obtain accurate results by such approaches. Second, the selectivity is related to the actual ion current and not directly to the individual activation barriers. Thus, it is essential to simulate the ion currents and this cannot be accomplished at present by microscopic MD approaches. In order to address this challenge, we developed and refined an approach capable of evaluating ion current while still reflecting the realistic features of the given channel. Our method involves generation of semimacroscopic free energy surfaces for the channel/ions system and Brownian dynamics (BD) simulations of the corresponding ion current. In contrast to most alternative macroscopic models, our approach is able to reproduce the difference between the free energy surfaces of different ions and thus to address the selectivity problem. Our method is used in a study of the selectivity of the KcsA channel toward the K+ and Na+ ions. The BD simulations with the calculated free energy profiles produce an appreciable selectivity. To the best of our knowledge, this is the first time that the trend in the selectivity in the ion current is produced by a computer simulation approach. Nevertheless, the calculated selectivity is still smaller than its experimental estimate. Recognizing that the calculated profiles are not perfect, we examine how changes in these profiles can account for the observed selectivity. It is found that the origin of the selectivity is more complex than generally assumed. The observed selectivity can be reproduced by increasing the barrier at the exit and the entrance of the selectivity filter, but the necessary changes in the barrier approach the limit of the error in the PDLD/S-LRA calculations. Other options that can increase the selectivity are also considered, including the difference between the Na+...Na+ and K+...K+ interaction. However, this interesting effect does not appear to lead to a major difference in selectivity since the Na+ ions at the limit of strong interaction tend to move in a less concerted way than the K+ ions. Changes in the relative binding energies at the different binding sites are also not so effective in changing the selectivity. Finally, it is pointed out that using the calculated profiles as a starting point and forcing the model to satisfy different experimentally based constraints, should eventually provide more detailed understanding of the different complex factors involved in ion selectivity of biological channels.     

Testing the applicability of Nernst-Planck theory in ion channels: comparisons with Brownian dynamics simulations

The macroscopic Nernst-Planck (NP) theory has often been used for predicting ion channel currents in recent years, but the validity of this theory at the microscopic scale has not been tested. In this study we systematically tested the ability of the NP theory to accurately predict channel currents by combining and comparing the results with those of Brownian dynamics (BD) simulations. To thoroughly test the theory in a range of situations, calculations were made in a series of simplified cylindrical channels with radii ranging from 3 to 15 Å, in a more complex ‘catenary’ channel, and in a realistic model of the mechanosensitive channel MscS. The extensive tests indicate that the NP equation is applicable in narrow ion channels provided that accurate concentrations and potentials can be input as the currents obtained from the combination of BD and NP match well with those obtained directly from BD simulations, although some discrepancies are seen when the ion concentrations are not radially uniform. This finding opens a door to utilising the results of microscopic simulations in continuum theory, something that is likely to be useful in the investigation of a range of biophysical and nano-scale applications and should stimulate further studies in this direction.     

A Grand Canonical Monte Carlo-Brownian dynamics algorithm for simulating ion channels

A computational algorithm based on Grand Canonical Monte Carlo (GCMC) and Brownian Dynamics (BD) is described to simulate the movement of ions in membrane channels. The proposed algorithm, GCMC/BD, allows the simulation of ion channels with a realistic implementation of boundary conditions of concentration and transmembrane potential. The method is consistent with a statistical mechanical formulation of the equilibrium properties of ion channels (; Biophys. J. 77:139-153). The GCMC/BD algorithm is illustrated with simulations of simple test systems and of the OmpF porin of Escherichia coli. The approach provides a framework for simulating ion permeation in the context of detailed microscopic models.     

Mechanisms of permeation and selectivity in calcium channels

The mechanisms underlying ion transport and selectivity in calcium channels are examined using electrostatic calculations and Brownian dynamics simulations. We model the channel as a rigid structure with fixed charges in the walls, representing glutamate residues thought to be responsible for ion selectivity. Potential energy profiles obtained from multi-ion electrostatic calculations provide insights into ion permeation and many other observed features of L-type calcium channels. These qualitative explanations are confirmed by the results of Brownian dynamics simulations, which closely reproduce several experimental observations. These include the current-voltage curves, current-concentration relationship, block of monovalent currents by divalent ions, the anomalous mole fraction effect between sodium and calcium ions, attenuation of calcium current by external sodium ions, and the effects of mutating glutamate residues in the amino acid sequence.     

Hierarchical approach to predicting permeation in ion channels

A hierarchical computational strategy combining molecular modeling, electrostatics calculations, molecular dynamics, and Brownian dynamics simulations is developed and implemented to compute electrophysiologically measurable properties of the KcsA potassium channel. Models for a series of channels with different pore sizes are developed from the known x-ray structure, using insights into the gating conformational changes as suggested by a variety of published experiments. Information on the pH dependence of the channel gating is incorporated into the calculation of potential profiles for K(+) ions inside the channel, which are then combined with K(+) ion mobilities inside the channel, as computed by molecular dynamics simulations, to provide inputs into Brownian dynamics simulations for computing ion fluxes. The open model structure has a conductance of approximately 110 pS under symmetric 250 mM K(+) conditions, in reasonable agreement with experiments for the largest conducting substate. The dimensions of this channel are consistent with electrophysiologically determined size dependence of quaternary ammonium ion blocking from the intracellular end of this channel as well as with direct structural evidence that tetrabutylammonium ions can enter into the interior cavity of the channel. Realistic values of Ussing flux ratio exponents, distribution of ions within the channel, and shapes of the current-voltage and current-concentration curves are obtained. The Brownian dynamics calculations suggest passage of ions through the selectivity filter proceeds by a "knock-off" mechanism involving three ions, as has been previously inferred from functional and structural studies of barium ion blocking. These results suggest that the present calculations capture the essential nature of K(+) ion permeation in the KcsA channel and provide a proof-of-concept for the integrated microscopic/mesoscopic multitiered approach for predicting ion channel function from structure, which can be applied to other channel structures.     

Ion permeation and selectivity of OmpF porin: a theoretical study based on molecular dynamics,Brownian dynamics,and continuum electrodiffusion theory

Three different theoretical approaches are used and compared to refine our understanding of ion permeation through the channel formed by OmpF porin from Escherichia coli. Those approaches are all-atom molecular dynamics (MD) in which ions, solvent, and lipids are represented explicitly, Brownian dynamics (BD) in which ions are represented explicitly, while solvent and lipids are represented as featureless dielectrics, and Poisson-Nernst-Planck (PNP) electrodiffusion theory in which both solvent and local ion concentrations are represented as a continuum. First, the ability of the different theoretical approaches in reproducing the equilibrium average ion density distribution in OmpF porin bathed by a 1M KCl symmetric salt solution is examined. Under those conditions the PNP theory is equivalent to the non-linear Poisson-Boltzmann (PB) theory. Analysis shows that all the three approaches are able to capture the important electrostatic interactions between ions and the charge distribution of the channel that govern ion permeation and selectivity in OmpF. The K(+) and Cl(-) density distributions obtained from the three approaches are very consistent with one another, which suggests that a treatment on the basis of a rigid protein and continuum dielectric solvent is valid in the case of OmpF. Interestingly, both BD and continuum electrostatics reproduce the distinct left-handed twisted ion pathways for K(+) and Cl(-) extending over the length of the pore which were observed previously in MD. Equilibrium BD simulations in the grand canonical ensemble indicate that the channel is very attractive for cations, particularly at low salt concentration. On an average there is 1.55 K(+) inside the pore in 10mM KCl. Remarkably, there is still 0.17 K(+) on average inside the pore even at a concentration as low as 1microM KCl. Secondly, non-equilibrium ion flow through OmpF is calculated using BD and PNP and compared with experimental data. The channel conductance in 0.2M and 1M KCl calculated using BD is in excellent accord with the experimental data. The calculations reproduce the experimentally well-known conductance-concentration relation and also reveal an asymmetry in the channel conductance (a larger conductance is observed under a positive transmembrane potential). Calculations of the channel conductance for three mutants (R168A, R132A, and K16A) in 1M KCl suggest that the asymmetry in the channel conductance arises mostly from the permanent charge distribution of the channel rather than the shape of the pore itself. Lastly, the calculated reversal potential in a tenfold salt gradient (0.1:1M KCl) is 27.4(+/-1.3)mV (BD) and 22.1(+/-0.6)mV (PNP), in excellent accord with the experimental value of 24.3mV. Although most of the results from PNP are qualitatively reasonable, the calculated channel conductance is about 50% higher than that calculated from BD probably because of a lack of some dynamical ion-ion correlations.     

Saxitoxin is a gating modifier of HERG K+ channels

Potassium (K+) channels mediate numerous electrical events in excitable cells, including cellular membrane potential repolarization. The hERG K+ channel plays an important role in myocardial repolarization, and inhibition of these K+ channels is associated with long QT syndromes that can cause fatal cardiac arrhythmias. In this study, we identify saxitoxin (STX) as a hERG channel modifier and investigate the mechanism using heterologous expression of the recombinant channel in HEK293 cells. In the presence of STX, channels opened slower during strong depolarizations, and they closed much faster upon repolarization, suggesting that toxin-bound channels can still open but are modified, and that STX does not simply block the ion conduction pore. STX decreased hERG K+ currents by stabilizing closed channel states visualized as shifts in the voltage dependence of channel opening to more depolarized membrane potentials. The concentration dependence for steady-state modification as well as the kinetics of onset and recovery indicate that multiple STX molecules bind to the channel. Rapid application of STX revealed an apparent "agonist-like" effect in which K+ currents were transiently increased. The mechanism of this effect was found to be an effect on the channel voltage-inactivation relationship. Because the kinetics of inactivation are rapid relative to activation for this channel, the increase in K+ current appeared quickly and could be subverted by a decrease in K+ currents due to the shift in the voltage-activation relationship at some membrane potentials. The results are consistent with a simple model in which STX binds to the hERG K+ channel at multiple sites and alters the energetics of channel gating by shifting both the voltage-inactivation and voltage-activation processes. The results suggest a novel extracellular mechanism for pharmacological manipulation of this channel through allosteric coupling to channel gating.     

Modification of cyclic nucleotide-gated ion channels by ultraviolet light

We irradiated cyclic nucleotide-gated ion channels in situ with ultraviolet light to probe the role of aromatic residues in ion channel function. UV light reduced the current through excised membrane patches from Xenopus oocytes expressing the alpha subunit of bovine retinal cyclic nucleotide-gated channels irreversibly, a result consistent with permanent covalent modification of channel amino acids by UV light. The magnitude of the current reduction depended only on the total photon dose delivered to the patches, and not on the intensity of the exciting light, indicating that the functionally important photochemical modification(s) occurred from an excited state reached by a one-photon absorption process. The wavelength dependence of the channels' UV light sensitivity (the action spectrum) was quantitatively consistent with the absorption spectrum of tryptophan, with a small component at long wavelengths, possibly due to cystine absorption. This spectral analysis suggests that UV light reduced the currents at most wavelengths studied by modifying one or more "target" tryptophans in the channels. Comparison of the channels' action spectrum to the absorption spectrum of tryptophan in various solvents suggests that the UV light targets are in a water-like chemical environment. Experiments on mutant channels indicated that the UV light sensitivity of wild-type channels was not conferred exclusively by any one of the 10 tryptophan residues in a subunit. The similarity in the dose dependences of channel current reduction and tryptophan photolysis in solution suggests that photochemical modification of a small number of tryptophan targets in the channels is sufficient to decrease the currents.     

A uniquely adaptable pore is consistent with NALCN being an ion sensor

NALCN is an intriguing, orphan ion channel among the 4x6TM family of related voltage-gated cation channels, sharing a common architecture of four homologous domains consisting of six transmembrane helices, separated by three cytoplasmic linkers and delimited by N and C-terminal ends. NALCN is one of the shortest 4x6TM family members, lacking much of the variation that provides the diverse palate of gating features, and tissue specific adaptations of sodium and calcium channels. NALCN’s most distinctive feature is that that it possesses a highly adaptable pore with a calcium-like EEEE selectivity filter in radially symmetrical animals and a more sodium-like EEKE or EKEE selectivity filter in bilaterally symmetrical animals including vertebrates. Two lineages of animals evolved alternative calcium-like EEEE and sodium-like EEKE / EKEE pores, spliced to regulate NALCN functions in differing cellular environments, such as muscle (heart and skeletal) and secretory tissue (brain and glands), respectively. A highly adaptable pore in an otherwise conserved ion channel in the 4x6TM channel family is not consistent with a role for NALCN in directly gating a significant ion conductance that can be either sodium ions or calcium ions. NALCN was proposed to be an expressible Gd³⁺-sensitive, NMDG⁺-impermeant, non-selective and ohmic leak conductance in HEK-293T cells, but we were unable to distinguish these reported currents from leaky patch currents (I_LP) in control HEK-293T cells. We suggest that NALCN functions as a sensor for the much larger UNC80/UNC79 complex, in a manner consistent with the coupling mechanism known for other weakly or non-conducting 4x6TM channel sensor proteins such as Na_x or Ca_v1.1. We propose that NALCN serves as a variable sensor that responds to calcium or sodium ion flux, depending on whether the total cellular current density is generated more from calcium-selective or sodium-selective channels.     

Invariant manifold reductions for Markovian ion channel dynamics

We show that many Markov models of ion channel kinetics have globally attracting stable invariant manifolds, even when the Markov process is time dependent. The primary implication of this is that, since the dimension of the invariant manifold is often substantially smaller than the full master equation system, simulations of ion channel kinetics can be substantially simplified, with no approximation. We show that this applies to certain models of potassium channels, sodium channels, ryanodine receptors and IP₃ receptors. We also use this to show that the original Hodgkin–Huxley formulations of potassium channel conductance and sodium channel conductance are the exact solutions of full Markov models for these channels.      

Statistical properties of ion channel records. Part II: estimation from the macroscopic current

Macroscopic ion channel current is the summation of the stochastic records of individual channel currents and therefore relates to their statistical properties. As a consequence of this relationship, it may be possible to derive certain statistical properties of single channel records or even generate some estimates of the records themselves from the macroscopic current when the direct measurement of single channel currents is not applicable. We present a procedure for generating the single channel records of an ion channel from its macroscopic current when the stochastic process of channel gating has the following two properties: (I) the open duration is independent of the time of opening event and has a single exponential probability density function (pdf), (II) all the channels have the same probability to open at time t. The application of this procedure is considered for cases where direct measurement of single channel records is difficult or impossible. First, the probability density function (pdf) of opening events, a statistical property of single channel records, is derived from the normalized macroscopic current and mean channel open duration. Second, it is shown that under the conditions (I) and (II), a non-stationary Markov model can represent the stochastic process of channel gating. Third, the non-stationary Markov model is calibrated using the results of the first step. The non-stationary formulation increases the model ability to generate a variety of different single channel records compared to common stationary Markov models. The model is then used to generate single channel records and to obtain other statistical properties of the records. Experimental single channel records of inactivating BK potassium channels are used to evaluate how accurately this procedure reconstructs measured single channel sweeps.     

Protons and Psalmotoxin-1 reveal nonproton ligand stimulatory sites in chicken acid-sensing ion channel

Acid-sensing ion channels (ASICs) are proton-sensitive, sodium-selective channels expressed in the nervous system that sense changes in extracellular pH. These ion channels are sensitive to an increasing number of nonproton ligands that include natural venom peptides and guanidine compounds. In the case of chicken ASIC1, the spider toxin Psalmotoxin-1 (PcTx1) activates the channel, resulting in an inward current. Furthermore, a growing class of ligands containing a guanidine group has been identified that stimulate peripheral ASICs (ASIC3), but exert subtle influence on other ASIC subtypes. The effects of the guanidine compounds on cASIC1 have not been the focus of previous study. Here, we investigated the interaction of the guanidine compound 2-guanidine-4-methylquinazoline (GMQ) on cASIC1 proton activation and PcTx1 stimulation. Exposure of expressed cASIC1 to PcTx1 resulted in biphasic currents consisting of a transient peak followed by an irreversible cASIC1 PcTx1 persistent current. This cASIC1 PcTx1 persistent current may be the result of locking the cASIC1 protein into a desensitized transition state. The guanidine compound GMQ increased the apparent affinity of protons on cASIC1 and decreased the half-maximal constant of the cASIC1 steady-state desensitization profile. Furthermore, GMQ stimulated the cASIC1 PcTx1 persistent current in a concentration-dependent manner, which resulted in a non-desensitizing inward current. Our data suggests that GMQ may have multiple sites within cASIC1 and may act as a “molecular wedge” that forces the PcTx1-desensitized ASIC into an open state. Our findings indicate that guanidine compounds, such as GMQ, may alter acid-sensing ion channel activity in combination with other stimuli, and that additional ASIC subtypes (along with ASIC3) may serve to sense and mediate signals from multiple stimuli.     

Permeation of ions across the potassium channel: brownian dynamics studies

The physical mechanisms underlying the transport of ions across a model potassium channel are described. The shape of the model channel corresponds closely to that deduced from crystallography. From electrostatic calculations, we show that an ion permeating the channel, in the absence of any residual charges, encounters an insurmountable energy barrier arising from induced surface charges. Carbonyl groups along the selectivity filter, helix dipoles near the oval chamber, and mouth dipoles near the channel entrances together transform the energy barrier into a deep energy well. Two ions are attracted to this well, and their presence in the channel permits ions to diffuse across it under the influence of an electric field. Using Brownian dynamics simulations, we determine the magnitude of currents flowing across the channel under various conditions. The conductance increases with increasing dipole strength and reaches its maximum rapidly; a further increase in dipole strength causes a steady decrease in the channel conductance. The current also decreases systematically when the effective dielectric constant of the channel is lowered. The conductance with the optimal choice of dipoles reproduces the experimental value when the dielectric constant of the channel is assumed to be 60. The current-voltage relationship obtained with symmetrical solutions is linear when the applied potential is less than approximately 100 mV but deviates from Ohm's law at a higher applied potential. The reversal potentials obtained with asymmetrical solutions are in agreement with those predicted by the Nernst equation. The conductance exhibits the saturation property observed experimentally. We discuss the implications of these findings for the transport of ions across the potassium channels and membrane channels in general.     

Identification and experimental verification of a novel family of bacterial cyclic nucleotide-gated (bCNG) ion channels

Studies of bacterial ion channels have provided significant insights into the structure-function relationships of mechanosensitive and voltage-gated ion channels. However, to date, very few bacterial channels that respond to small molecules have been identified, cloned, and characterized. Here, we use bioinformatics to identify a novel family of bacterial cyclic nucleotide-gated (bCNG) ion channels containing a channel domain related by sequence homology to the mechanosensitive channel of small conductance (MscS). In this initial report, we clone selected members of this channel family, use electrophysiological measurements to verify their ability to directly gate in response to cyclic nucleotides, and use osmotic downshock to demonstrate their lack of mechanosensitivity. In addition to providing insight into bacterial physiology, these channels will provide researchers with a useful model system to investigate the role of ligand-gated ion channels (LGICs) in the signaling processes of higher organisms. The identification of these channels provides a foundation for structural and functional studies of LGICs that would be difficult to perform on mammalian channels. Moreover, the discovery of bCNG channels implies that bacteria have cyclic nucleotide-gated and cyclic nucleotide-modulated ion channels, which are analogous to the ion channels involved in eukaryotic secondary messenger signaling pathways.     

Protons and Psalmotoxin-1 reveal nonproton ligand stimulatory sites in chicken acid-sensing ion channel: Implication for simultaneous modulation in ASICs

Acid-sensing ion channels (ASICs) are proton-sensitive, sodium-selective channels expressed in the nervous system that sense changes in extracellular pH. These ion channels are sensitive to an increasing number of nonproton ligands that include natural venom peptides and guanidine compounds. In the case of chicken ASIC1, the spider toxin Psalmotoxin-1 (PcTx1) activates the channel, resulting in an inward current. Furthermore, a growing class of ligands containing a guanidine group has been identified that stimulate peripheral ASICs (ASIC3), but exert subtle influence on other ASIC subtypes. The effects of the guanidine compounds on cASIC1 have not been the focus of previous study. Here, we investigated the interaction of the guanidine compound 2-guanidine-4-methylquinazoline (GMQ) on cASIC1 proton activation and PcTx1 stimulation. Exposure of expressed cASIC1 to PcTx1 resulted in biphasic currents consisting of a transient peak followed by an irreversible cASIC1 PcTx1 persistent current. This cASIC1 PcTx1 persistent current may be the result of locking the cASIC1 protein into a desensitized transition state. The guanidine compound GMQ increased the apparent affinity of protons on cASIC1 and decreased the half-maximal constant of the cASIC1 steady-state desensitization profile. Furthermore, GMQ stimulated the cASIC1 PcTx1 persistent current in a concentration-dependent manner, which resulted in a non-desensitizing inward current. Our data suggests that GMQ may have multiple sites within cASIC1 and may act as a “molecular wedge” that forces the PcTx1-desensitized ASIC into an open state. Our findings indicate that guanidine compounds, such as GMQ, may alter acid-sensing ion channel activity in combination with other stimuli, and that additional ASIC subtypes (along with ASIC3) may serve to sense and mediate signals from multiple stimuli.     

An electrostatic model of a membrane ion pump

The model is based upon an ion channel with an electric dipolar structure. With simplifying assumptions it is possible to calculate that a typical channel, 1 nm in diameter and 5 nm long, could contain at most two or three univalent cations at a time. The channel ion binding sites have an effective affinity for ions from the fluid bathing the negative end of the channel, several orders of magnitude higher than their affinity for ions from the fluid bathing the positive end of the channel. The approach of an external, positively charged body to the negative end of the channel, is sufficient to convert the two- or three-channel ion sites with high affinity for ions from the fluid bathing this end into very low affinity sites for the same ions that now have access only to the fluid bathing the other end of the channel. The change in affinity and fluid access requires no molecular or electrical change in the channel structure other than the passive superposition of the electrostatic potential of the dipolar channel and that of the charged body. An oscillating electric field externally applied to an electric dipolar channel is shown to result in the unidirectional pumping of cations in the direction of the channel dipole even against large adverse ion concentration gradients. The energy required must be supplied by the sources of the electric field. By using two such channels in close proximity, one selective for K+ ions with its dipole moment pointing into a cell and the other selective for Na+ ions with its dipole moment pointing out from the cell, it is possible to construct a model pump with calculated properties that simulate many of those measured for Na+-K+-ATPase, with both physiological and artificial ionic concentrations.     

Membranes with the same ion channel populations but different excitabilities

Electrical signaling allows communication within and between different tissues and is necessary for the survival of multicellular organisms. The ionic transport that underlies transmembrane currents in cells is mediated by transporters and channels. Fast ionic transport through channels is typically modeled with a conductance-based formulation that describes current in terms of electrical drift without diffusion. In contrast, currents written in terms of drift and diffusion are not as widely used in the literature in spite of being more realistic and capable of displaying experimentally observable phenomena that conductance-based models cannot reproduce (e.g. rectification). The two formulations are mathematically related: conductance-based currents are linear approximations of drift-diffusion currents. However, conductance-based models of membrane potential are not first-order approximations of drift-diffusion models. Bifurcation analysis and numerical simulations show that the two approaches predict qualitatively and quantitatively different behaviors in the dynamics of membrane potential. For instance, two neuronal membrane models with identical populations of ion channels, one written with conductance-based currents, the other with drift-diffusion currents, undergo transitions into and out of repetitive oscillations through different mechanisms and for different levels of stimulation. These differences in excitability are observed in response to excitatory synaptic input, and across different levels of ion channel expression. In general, the electrophysiological profiles of membranes modeled with drift-diffusion and conductance-based models having identical ion channel populations are different, potentially causing the input-output and computational properties of networks constructed with these models to be different as well. The drift-diffusion formulation is thus proposed as a theoretical improvement over conductance-based models that may lead to more accurate predictions and interpretations of experimental data at the single cell and network levels.     

Ion behavior in the selectivity filter of HCN1 channels

Hyperpolarization-activated cyclic-nucleotide gated channels (HCNs) are responsible for the generation of pacemaker currents (I_f or I_h) in cardiac and neuronal cells. Despite the overall structural similarity to voltage-gated potassium (Kv) channels, HCNs show much lower selectivity for K⁺ over Na⁺ ions. This increased permeability to Na⁺ is critical to their role in membrane depolarization. HCNs can also select between Na⁺ and Li⁺ ions. Here, we investigate the unique ion selectivity properties of HCNs using molecular-dynamics simulations. Our simulations suggest that the HCN1 pore is flexible and dilated compared with Kv channels with only one stable ion binding site within the selectivity filter. We also observe that ion coordination and hydration differ within the HCN1 selectivity filter compared with those in Kv and cyclic-nucleotide gated channels. Additionally, the C358T mutation further stabilizes the symmetry of the binding site and provides a more fit space for ion coordination, particularly for Li⁺.