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Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Structural analysis of CFTR has identified a narrow, hydrophobic region close to the extracellular end of the open channel pore that may function as a selectivity filter. The present study combines comprehensive mutagenesis of hydrophobic amino-acid side-chains within the selectivity filter with functional evaluation of channel Cl− conductance and anion selectivity. Among these hydrophobic amino-acids, one (F337) appears to play a dominant role in determining both conductance and selectivity. Anion selectivity appears to depend on both side-chain size and hydrophobicity at this position. In contrast, conductance is disrupted by all F337 mutations, suggesting that unique interactions between permeating Cl− ions and the native phenylalanine side-chain are important for conductance. Surprisingly, a positively charged lysine side-chain can be substituted for several hydrophobic residues within the selectivity filter (including F337) with only minor changes in pore function, arguing against a crucial role for overall hydrophobicity. These results suggest that localized interactions between permeating anions and amino-acid side-chains within the selectivity filter may be more important in determining pore functional properties than are global features such as overall hydrophobicity. [ABSTRACT FROM AUTHOR]

Developing environmentally friendly and inexpensive membranes for water purifying applications is a global challenge. Graphene oxide is one of the most-studied and tailorable materials for membrane fabrication; however, adjustment of interlayer spacings for an efficient desalination performance has not yet been fully achieved. Herein, graphene oxide was reduced with ascorbic acid for 5–180 min. The partially reduced graphene oxide was utilized through vacuum filtration for membrane preparation. Reduced samples for 5–20 min could form intact membranes, which showed excellent water flux (58–178 L m−2 h−1 bar−1) and efficiently reduced NaCl permeability. Besides, the partially reduced graphene oxides revealed dual interlayer spacings related to almost graphitic domains and semi-reduced domains comprising oxygen-containing functional groups. By investigating the water flow and ion permeation mechanism, the remaining oxidized domains and the major types of functional groups were found to be modulating, respectively. This study showed that partial reduction could be facilely exploited to fabricate graphene oxide-based membranes with adjustable interlayer spacing capable of salt blocking. [ABSTRACT FROM AUTHOR]

P2X receptor channels are trimeric ATP-activated ion channels expressed in neuronal and non-neuronal cells that are attractive therapeutic targets for human disorders. Seven subtypes of P2X receptor channels have been identified in mammals that can form both homomeric and heteromeric channels. P2X1–4 and P2X7 receptor channels are cation-selective, whereas P2X5 has been reported to have both cation and anion permeability. P2X receptor channel structures reveal that each subunit is comprised of two transmembrane helices, with both N-and C-termini on the intracellular side of the membrane and a large extracellular domain that contains the ATP binding sites at subunit interfaces. Recent structures of ATP-bound P2X receptors with the activation gate open reveal the unanticipated presence of a cytoplasmic cap over the central ion permeation pathway, leaving lateral fenestrations that may be largely buried within the membrane as potential pathways for ions to permeate the intracellular end of the pore. In the present study, we identify a critical residue within the intracellular lateral fenestrations that is readily accessible to thiol-reactive compounds from both sides of the membrane and where substitutions influence the relative permeability of the channel to cations and anions. Taken together, our results demonstrate that ions can enter or exit the internal pore through lateral fenestrations that play a critical role in determining the ion selectivity of P2X receptor channels.

Potassium (K + ) channels combine high conductance with high ion selectivity. To explain this efficiency, two molecular mechanisms have been proposed. The "direct knock-on" mechanism is defined by water-free K + permeation and formation of direct ion-ion contacts in the highly conserved selectivity filter (SF). The "soft knock-on" mechanism involves co-permeation of water and separation of K + by water molecules. With the aim to distinguish between these mechanisms, crystal structures of the KcsA channel with mutations in two SF residues-G77 and T75-were published, where the arrangements of K + ions and water display canonical soft knock-on configurations. These data were interpreted as evidence of the soft knock-on mechanism in wild-type channels. Here, we test this interpretation using molecular dynamics simulations of KcsA and its mutants. We show that while a strictly water-free direct knock-on permeation is observed in the wild type, conformational changes induced by these mutations lead to distinct ion permeation mechanisms, characterized by co-permeation of K + and water. These mechanisms are characterized by reduced conductance and impaired potassium selectivity, supporting the importance of full dehydration of potassium ions for the hallmark high conductance and selectivity of K + channels. In general, we present a case where mutations introduced at the critical points of the permeation pathway in an ion channel drastically change its permeation mechanism in a nonintuitive manner., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel shows only weak selectivity between different small monovalent anions, however, little is known about its ability to discriminate between monovalent and divalent anions. The present study uses patch clamp recording to investigate the interaction between the small divalent anions S2O32− and SO42− and wild-type and pore-mutant forms of human CFTR. Binding of these anions to wild-type CFTR appears weak; at 10 mM, intracellular S2O32− and SO42− blocked <20 and <5% of macroscopic Cl− current respectively, while these same concentrations had no discernible blocking effect when present in the extracellular solution. However, introduction of additional positive charge into the inner vestibule of the pore (in I344K and S1141K mutant channels) drastically strengthened block by intracellular (but not extracellular) S2O32− and SO42−. Block of these mutant channels was highly voltage-dependent; at very negative membrane potentials, apparent binding affinities were ~100 µM for S2O32− and <1 mM for SO42−. Permeability of S2O32− and SO42− was too small to be quantified in wild-type CFTR, but was <1% of Cl− permeability. Mutants that strengthened divalent binding (I344K, S1141K), as well as the selectivity-altering mutant F337A, also showed immeasurably low S2O32− and SO42− permeabilities. Overall CFTR selects well for monovalent over divalent anions, both in terms of binding and permeability. The number or density of fixed positive charges in the pore appears well optimized to disfavour binding of divalent anions, which may be an important facet of the monovalent Cl− permeation mechanism. [ABSTRACT FROM AUTHOR]

Sodium channels selectively conduct Na+ ions across cellular membrane with extraordinary efficiency, which is essential for initiating action potentials. However, how Na+ ions permeate the ionic channels remains obscure and ambiguous. With more than 40 conductance events from microsecond molecular dynamics simulation, the soft knock-on ion permeation mediated by water molecules was observed and confirmed by the free energy profile and electrostatic potential calculation in this study. During the soft knock-on process, the change of average distance between four oxygen atoms in Glu177-Glu177 plays a very important role for the permeation of Na+ ion. Exploration of the ionic conductance mechanism could provide a guideline for designing ion channel targeted drug. [ABSTRACT FROM AUTHOR]

Prokaryotic channels play an important role in the structural biology of ion channels. At the end of the 20th century, the first structure of a prokaryotic ion channel was revealed. Subsequently, the reporting of structures of various prokaryotic ion channels have provided fundamental insights into the structure of ion channels of higher organisms. Voltage-dependent Ca2+ channels (Cavs) are indispensable for coupling action potentials with Ca2+ signaling. Similar to other proteins, Cavs were predicted to have a prokaryotic counterpart; however, it has taken more than 20 years for one to be identified. The homotetrameric channel obtained from Meiothermus ruber generates the calcium ion specific current, so it is named as CavMr. Its selectivity filter contains a smaller number of negatively charged residues than mutant Cavs generated from other prokaryotic channels. CavMr belonged to a different cluster of phylogenetic trees than canonical prokaryotic cation channels. The glycine residue of the CavMr selectivity filter is a determinant for calcium selectivity. This glycine residue is conserved among eukaryotic Cavs, suggesting that there is a universal mechanism for calcium selectivity. A family of homotetrameric channels has also been identified from eukaryotic unicellular algae, and the investigation of these channels can help to understand the mechanism for ion selection that is conserved from prokaryotes to eukaryotes.

Cyclic nucleotide–gated (CNG) channels are key to the signal transduction machinery of certain sensory modalities both in vertebrate and invertebrate organisms. They translate a chemical change in cyclic nucleotide concentration into an electrical signal that can spread through sensory cells. Despite CNG and voltage-gated potassium channels sharing a remarkable amino acid sequence homology and basic architectural plan, their functional properties are dramatically different. While voltage-gated potassium channels are highly selective and require membrane depolarization to open, CNG channels have low ion selectivity and are not very sensitive to voltage. In the last few years, many high-resolution structures of intact CNG channels have been released. This wealth of new structural information has provided enormous progress toward the understanding of the molecular mechanisms and driving forces underpinning CNG channel activation. In this review, we report on the current understanding and controversies surrounding the gating mechanism in CNG channels, as well as the deep intertwining existing between gating, the ion permeation process, and its modulation by membrane voltage. While the existence of this powerful coupling was recognized many decades ago, its direct structural demonstration, and ties to the CNG channel inherent pore flexibility, is a recent achievement. [ABSTRACT FROM AUTHOR]

The P2X7 receptor, originally known as the P2Z receptor due to its distinctive functional properties, has a structure characteristic of the ATP-gated ion channel P2X receptor family. The P2X7 receptor is an important mediator of ATP-induced purinergic signalling and is involved the pathogenesis of numerous conditions as well as in the regulation of diverse physiological functions. Functional characterisations, in conjunction with site-directed mutagenesis, molecular modelling, and, recently, structural determination, have provided significant insights into the structure–function relationships of the P2X7 receptor. This review discusses the current understanding of the structural basis for the functional properties of the P2X7 receptor. [ABSTRACT FROM AUTHOR]

The structural stability and high permeability of 2D materials‐based membranes are key research interests for separation membranes. However, the instability of their microstructures due to the aqueous‐induced swelling phenomenon affects their performance. It is highly desirable to explore anti‐swelling approaches able to mitigate this challenge as well as develop 2D lamellar membranes with ion selective capabilities beyond the limits based on size exclusion mechanism. Herein, graphene oxide (GO) laminate membranes covalently anchored and intercalated with metal‐phenolic networks display suppressed swelling durability and resist delamination in aqueous media. The resultant Fe@TA‐GO membrane with confined d‐spacing boasts high water fluxes of up to 15.0 L m−2 h−1 under osmotic pressure (2 m sucrose). Furthermore, the permeation tests reveal that the interaction between the metal ions and the immobilized cation–ligand molecules influences the ion permeances through the functionalized GO membranes conducing to fast ion permeation rates with ion diffusion permselectivity as well as effective selective separation of organic contaminants. These results will help advance the applications of GO membranes for challenging ion separations related to sustainable resource recovery and environmental remediation of organic dye and metal ion contaminated wastewaters. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels (Na(V)s) provide the initial electrical signal that drives action potential generation in many excitable cells of the brain, heart, and nervous system. For more than 60years, functional studies of Na(V)s have occupied a central place in physiological and biophysical investigation of the molecular basis of excitability. Recently, structural studies of members of a large family of bacterial voltage-gated sodium channels (BacNa(V)s) prevalent in soil, marine, and salt lake environments that bear many of the core features of eukaryotic Na(V)s have reframed ideas for voltage-gated channel function, ion selectivity, and pharmacology. Here, we analyze the recent advances, unanswered questions, and potential of BacNa(V)s as templates for drug development efforts.

[Display omitted] • The properties of C O groups in K+ channel could be regulated by THz waves. • The frequencies of 53 THz and 54 THz show as the inhibitory mode. • The frequencies of 53.6 THz and 53.75 THz show as the excitatory mode. • The excitatory point of 53.6 THz could increase the ion permeability and selectivity efficiency. The optical excitation effects offer an opportunity to gain insights into the structure and the function of K+ channel, contributing to the prediction of possible targets for drug design and precision therapy. Although there has been increasing research attention on the modulation of ion permeation in K+ channel by terahertz electromagnetic (THz-EM) stimuli, little exploration has been conducted regarding the dependence of ion permeation on frequencies. By using two-dimensional (2D) infrared excitation spectrum calculation for the K+ channel, we have discovered that the frequency of 53.60 THz serves as an optimal excitation modulation mode. This mode leads to an almost twofold enhancement in the rate of K+ ion permeation and a tenfold increase in selectivity efficiency. These improvements can be attributed to the coupling mode matching of the excited properties of C O groups in the K+ channel. Our findings propose a promising application of terahertz technology to improve the performance of ion channels, nanomembrane sieves, nanodevices, as well as neural therapy. [ABSTRACT FROM AUTHOR]

The importance of the microstzructure of silicone hydrogels is widely appreciated but is poorly understood and minimally investigated. To ensure comfort and eye health, these materials must simultaneously exhibit both high oxygen and high water permeability. In contrast with most conventional hydrogels, the water content and water structuring within silicone hydrogels cannot be solely used to predict permeability. The materials achieve these opposing requirements based on a composite of nanoscale domains of oxygen‐permeable (silicone) and water‐permeable hydrophilic components. This study correlated characteristic ion permeation coefficients of a selection of commercially available silicone hydrogel contact lenses with their morphological structure and chemical composition. Differential scanning calorimetry measured the water structuring properties through subdivision of the freezing water component into polymer‐associated water (loosely bound to the polymer matrix) and ice‐like water (unimpeded with a melting point close to that of pure water). Small‐angle x‐ray scattering, and environmental scanning electron microscopy techniques were used to investigate the structural morphology of the materials over a range of length scales. Significant, and previously unrecognized, differences in morphology between individual materials at nanometer length scales were determined; this will aid the design and performance of the next generation of ocular biomaterials, capable of maintaining ocular homeostasis. [ABSTRACT FROM AUTHOR]

[Display omitted] • Operating conditions of brine electrolyzer to produce NaOH and HCl are optimized. • Trade-off between reaction rate and caustic efficiency is addressed simultaneously. • The trade-off issue is solved by optimizing temperature and applied voltage. • The mechanism for performance improvement of the electrolyzer is revealed. • Long term stability test of the electrolyzer is conducted for 1600 h. Brine electrolyzers can produce HCl and NaOH concurrently using NaCl-concentrated wastewater discharged from various desalination processes. However, the electrolyzers still have poor performance, and thus commercialization is retarded. The main factor affecting the performance of this technology is the trade-off between reaction rate and caustic efficiency. This study aims to improve the reaction rate and caustic efficiency simultaneously by optimizing the operating conditions of the electrolyzer. Through various experiments, we have found that increasing the applied voltage and operating temperature can effectively enhance the reaction rate and caustic efficiency. This is because the relative permeation rate of sodium ions over protons through the Nafion membrane increases with temperature and applied voltage. Under the optimal conditions, the reaction rate is significantly enhanced by 2.1 times (from 86.5 mA cm−2 to 179.2 mA cm−2) and the caustic efficiency by 1.6 times (from 56% to 90%) compared to the standard conditions. The mechanism for the improved performance is revealed by investigating the permeation behavior of ions through the membrane. It is found that protons hinder the permeation of sodium ions, but increasing the temperature and/or applied voltage overcomes this interference. A long-term test for 1600 h confirms the stability and feasibility of the electrolyzer. Overall, this study is expected to contribute to commercializing brine electrolyzers for HCl and NaOH production. [ABSTRACT FROM AUTHOR]

Here, we present the atomic resolution crystallographic structure, the function, and the ion-binding properties of the KcsA mutants, G77A and G77C, that stabilize the 2,4-ion–bound configuration (i.e., water, K+, water, K+-ion–bound configuration) of the K+ channel’s selectivity filter. A full functional and thermodynamic characterization of the G77A mutant revealed wild-type–like ion selectivity and apparent K+-binding affinity, in addition to showing a lack of C-type inactivation gating and a marked reduction in its single-channel conductance. These structures validate, from a structural point of view, the notion that 2 isoenergetic ion-bound configurations coexist within a K+ channel’s selectivity filter, which fully agrees with the water–K+-ion–coupled transport detected by streaming potential measurements. [ABSTRACT FROM AUTHOR]

The evolutionarily conserved Piezo proteins, including Piezo1 and Piezo2, constitute a bona fide class of mechanosensitive (MS) cation channels, which play critical roles in various mammalian physiologies, including sensation of touch, proprioception and regulation of vascular development, and blood pressure. Furthermore, mutations in Piezos have been linked to various human genetic diseases, validating their potential as therapeutic targets. Thus, it is pivotal to understand how Piezo channels effectively convert mechanical force into selective cation permeation, and therefore precisely control the various mechanotransduction processes. On the basis of our recently determined cryoelectron microscopy structures of the full‐length 2547‐residue mouse Piezo1, structure‐guided mutagenesis, and electrophysiological and pharmacological characterizations, here we focus on reviewing the key structural features and functional components that enable Piezo1 to employ a lever‐like mechanogating mechanism to function as a sophisticated mechanotransduction channel. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels are the molecular components of electrical signaling in the body, yet the molecular origins of Na+-selective transport remain obscured by diverse protein chemistries within this family of ion channels. In particular, bacterial and mammalian sodium channels are known to exhibit similar relative ion permeabilities for Na+ over K+ ions, despite their distinct signature EEEE and DEKA sequences. Atomic-level molecular dynamics simulations using high-resolution bacterial channel structures and mammalian channel models have begun to describe how these sequences lead to analogous high field strength ion binding sites that drive Na+ conduction. Similar complexes have also been identified in unrelated acid sensing ion channels involving glutamate and aspartate side chains that control their selectivity. These studies suggest the possibility of a common origin for Na+ selective binding and transport. [ABSTRACT FROM AUTHOR]

Graphical abstract Fig. 1 Structures of TRPM channels. (A) Cartoon representation of a nTRPM2 (n: Nematostella vectensis) protomer. (B) Cartoon representation of a h TRPM4 (h: Homo sapiens) protomer. (C) Cartoon representation of a m TRPM7 (m: Mus musculus) protomer. (D) Cartoon representation of a f TRPM8 (f: Ficedula albicollis) protomer. (E) Comparison of n TRPM2 (green), h TRPM4 (cyan), m TRPM7 (magentas) and f TRPM8 (slate). Highlights • TRPM channels play a significant role in cell development and relevant • diseases. • Members of TRPMs display considerable structural and functional homology. • There are now four unique TRPM structures, which have presented many unprecedented structural findings. • The comparison between each new structure of TRPM channels brings many new biological insights. Abstract TRPM family (Transient receptor potential channels, M for melastatin) is a group of intrinsic plasma membrane ion channels which are widely expressed throughout human body. It has been identified as a potent entry point of working desperate diseases out in a new way with newfangled ideas and safer technological means. In our review, we discussed the common and unique properties of TRPM family with the elaborate narrate in their overall structures, different states and the underlying activation mechanism. Thus, this review can help to consummate the limited work of TRPM family and provide novel therapeutic targets of certain diseases. [ABSTRACT FROM AUTHOR]

We propose single-atom catalytic oxidation method for production of subnanometer-scale windows on the graphene layer of single walled carbon nanotubes (SWCNTs) in this study. We used the free-standing SWCNT films which had merits for reliable measurements and applicability to electrochemical measurements for determination of the nanowindow size. Copper(II) 2,3,9,10,16,17,23,24-octakis(octyloxy)-29 H ,31 H -phthalocyanine (CuPc) was adsorbed on SWCNT bundles using liquid-phase adsorption at surface coverage of 0.9. Then, nanowindows were produced by oxidation above 550 K being higher than the decomposition temperature of the bulk CuPc. The oxidation of CuPc-adsorbed SWCNT film markedly increased the surface area from N 2 adsorption at 77 K due to access of N 2 to the internal tube space through the nanowindows. The nanowindow size was estimated to be the molecular size of N 2 through the evident low-pressure adsorption hysteresis of the N 2 adsorption isotherm and TEM observation. The selective ion permeability through the nanowindows for H 3 O+, Li+, Na+, K+, Mg2+, Ca2+, and Ba2+ ions was determined by the inside-capacitance being the capacitance difference of SWCNT between with and without nanowindows. The selective permeation of the hydrated ions depended on the oxidation conditions for production of nanowindows, showing less than 1 nm of the nanowindow size. The relationship between the inside-capacitance and hydrated ion diameter for the selective permeation of hydrated ions indicated partial dehydration of the hydrated ions just only on their permeation through the nanowindows under the accelerated conditions. [Display omitted] [ABSTRACT FROM AUTHOR]

The permeability of large cations through the P2X pore has remained arguably the most controversial and complicated topic in P2X-related research, with the emergence of conflicting studies on the existence, mechanism and physiological relevance of a so-called “dilated” state. Due to the important role of several “dilating” P2X subtypes in numerous diseases, a clear and detailed understanding of this phenomenon represents a research priority. Recent advances, however, have challenged the existence of a progressive, ATP-induced pore dilation, by demonstrating that this phenomenon is an artifact of the method employed. Here, we discuss briefly the history of this controversial and enigmatic dilated state, from its initial discovery to its recent reconsideration. We will discuss the literature in which mechanistic pathways to a large cation-permeable state are proposed, as well as important advances in the methodology employed to study this elusive state. Considering recent literature, we will also open the discussion as to whether an intrinsically dilating P2X pore exists, as well as the physiological relevance of such a large cation-permeable pore and its potential use as therapeutic pathway.

The hair cell mechanotransduction (MET) channel complex is essential for hearing, yet it’s molecular identity and structure remain elusive. The transmembrane channel–like 1 (TMC1) protein localizes to the site of the MET channel, interacts with the tip-link responsible for mechanical gating, and genetic alterations in TMC1 alter MET channel properties and cause deafness, supporting the hypothesis that TMC1 forms the MET channel. We generated a model of TMC1 based on X-ray and cryo-EM structures of TMEM16 proteins, revealing the presence of a large cavity near the protein-lipid interface that also harbors the Beethoven mutation, suggesting that it could function as a permeation pathway. We also find that hair cells are permeable to 3 kDa dextrans, and that dextran permeation requires TMC1/2 proteins and functional MET channels, supporting the presence of a large permeation pathway and the hypothesis that TMC1 is a pore forming subunit of the MET channel complex.

Mous et al. recently reported the molecular mechanism of chloride transport through a light-activated pumping rhodopsin, a key process involved in a range of cellular functions. Their results open exciting new challenges for photopharmacology and computational modeling that should be addressed in the coming years. [ABSTRACT FROM AUTHOR]

The permeability of large cations through the P2X pore has remained arguably the most controversial and complicated topic in P2X-related research, with the emergence of conflicting studies on the existence, mechanism and physiological relevance of a so-called “dilated” state. Due to the important role of several “dilating” P2X subtypes in numerous diseases, a clear and detailed understanding of this phenomenon represents a research priority. Recent advances, however, have challenged the existence of a progressive, ATP-induced pore dilation, by demonstrating that this phenomenon is an artifact of the method employed. Here, we discuss briefly the history of this controversial and enigmatic dilated state, from its initial discovery to its recent reconsideration. We will discuss the literature in which mechanistic pathways to a large cation-permeable state are proposed, as well as important advances in the methodology employed to study this elusive state. Considering recent literature, we will also open the discussion as to whether an intrinsically dilating P2X pore exists, as well as the physiological relevance of such a large cation-permeable pore and its potential use as therapeutic pathway. [ABSTRACT FROM AUTHOR]

Abstract: The evolutionarily conserved Piezo family of proteins, including Piezo1 and Piezo2, encodes the long‐sought‐after mammalian mechanosensitive cation channels that play critical roles in various mechanotransduction processes such as touch, pain, proprioception, vascular development and blood pressure regulation. Mammalian Piezo proteins contain over 2500 amino acids with numerous predicted transmembrane segments, and do not bear sequence homology with any known class of ion channels. Thus, it is imperative, but challenging, to understand how they serve as effective mechanotransducers for converting mechanical force into electrochemical signals. Here, we review the recent major breakthroughs in determining the three‐bladed, propeller‐shaped structure of mouse Piezo1 using the state‐of‐the‐art cryo‐electron microscopy (cryo‐EM) and functionally dissecting out the molecular bases that define its ion permeation and mechanotransduction properties, which provide key insights into clarifying its oligomeric status and pore‐forming region. We also discuss the hypothesis that the complex Piezo proteins can be deduced into discrete mechanotransduction and ion‐conducting pore modules, which coordinate to fulfil their specialized function in mechanical sensing and transduction, ion permeation and selection. [ABSTRACT FROM AUTHOR]

This article reviews work presented at the ESCBM 2017 on using two-dimensional infrared (2D IR) spectroscopy to probe ion binding configurations in the potassium ion channel KcsA. We discuss two studies in which we use 2D IR spectroscopy in conjunction with protein semisynthesis and molecular dynamics (MD) simulations to test against two competing models of ion permeation and to investigate how changes in the conformation of the intracellular gate affect the structure and ion affinities of the selectivity filter. In our initial study, the 2D IR spectrum of an isotopically labeled KcsA filter reveals two spectral features that correspond to different structures and ion binding configurations. MD simulations help us link the experiments to atomistic structures, allowing us to determine the prevailing mechanism of ion conduction. In a follow-up study, we probe how ion occupancies in the filter change with the opening and closing of the intracellular gate. We experimentally modulate the conformation of KcsA using different sample conditions, and are able to show that the ion occupancies in the filter change with the state of the gate, revealing complexities in the conformational landscape of the potassium ion channel. Our work shows the potential of these techniques in addressing the fundamental biophysical questions in membrane protein structure and function. [ABSTRACT FROM AUTHOR]

Ion permeation through ion channels is essential for life. Since the determination of the x-ray crystal structure of the K+ channel, extensive molecular dynamics (MD) studies have been performed to visualize ion permeation through the channel. However, the mechanism underlying how the experimentally measured current amplitudes are determined still remains elusive. Here, we examined the MD-simulated trajectories of ion permeation through the Kv1.2 channel by developing a method analyzing the dynamics of ions. The analysis clarified that the entrance of ions into the channel determines the permeation rate. [ABSTRACT FROM AUTHOR]

This article reviews work presented at the ESCBM 2017 on using two-dimensional infrared (2D IR) spectroscopy to probe ion binding configurations in the potassium ion channel KcsA. We discuss two studies in which we use 2D IR spectroscopy in conjunction with protein semisynthesis and molecular dynamics (MD) simulations to test against two competing models of ion permeation and to investigate how changes in the conformation of the intracellular gate affect the structure and ion affinities of the selectivity filter. In our initial study, the 2D IR spectrum of an isotopically labeled KcsA filter reveals two spectral features that correspond to different structures and ion binding configurations. MD simulations help us link the experiments to atomistic structures, allowing us to determine the prevailing mechanism of ion conduction. In a follow-up study, we probe how ion occupancies in the filter change with the opening and closing of the intracellular gate. We experimentally modulate the conformation of KcsA using different sample conditions, and are able to show that the ion occupancies in the filter change with the state of the gate, revealing complexities in the conformational landscape of the potassium ion channel. Our work shows the potential of these techniques in addressing the fundamental biophysical questions in membrane protein structure and function. [ABSTRACT FROM AUTHOR]

Cryo-electron microscopy reveals the structure of a chloride channel that is closely related to a protein that transports lipids.

The calcium-activated chloride channel TMEM16A is a member of a conserved protein family that comprises ion channels and lipid scramblases. Although the structure of the scramblase nhTMEM16 has defined the architecture of the family, it was unknown how a channel has adapted to cope with its distinct functional properties. Here we have addressed this question by the structure determination of mouse TMEM16A by cryo-electron microscopy and a complementary functional characterization. The protein shows a similar organization to nhTMEM16, except for changes at the site of catalysis. There, the conformation of transmembrane helices constituting a membrane-spanning furrow that provides a path for lipids in scramblases has changed to form an enclosed aqueous pore that is largely shielded from the membrane. Our study thus reveals the structural basis of anion conduction in a TMEM16 channel and it defines the foundation for the diverse functional behavior in the TMEM16 family.

Increased extracellular proton concentrations during neurotransmission are converted to excitatory sodium influx by acid-sensing ion channels (ASICs). 10-fold sodium/potassium selectivity in ASICs has long been attributed to a central constriction in the channel pore, but experimental verification is lacking due to the sensitivity of this structure to conventional manipulations. Here, we explored the basis for ion selectivity by incorporating unnatural amino acids into the channel, engineering channel stoichiometry and performing free energy simulations. We observed no preference for sodium at the “GAS belt” in the central constriction. Instead, we identified a band of glutamate and aspartate side chains at the lower end of the pore that enables preferential sodium conduction.

Inorganic ions are critical for cellular function and require an efficient mechanism of transport through the cellular membrane. Most often the transport of ions occurs through proteins known as ion channels and transporters. Ion binding and permeation through these proteins is a complicated process that is still under investigation with a wide range of experimental and theoretical methods. Here we present an overview of some of the competing theories of ion transport with special emphasis on the theoretical methods used for the elucidation of the energetics of ion selectivity, coordination and permeation. A large part of the review is dedicated to potassium and sodium channels and transporters, which are among the best studied biological transport systems and provide a frame of reference for all other ion-protein interactions. In addition, we summarize the computational work done on the transport of several other small inorganic ions (calcium, magnesium, chloride, inorganic phosphate). Our aim is to provide a general picture of the current state of knowledge on biological ion-transport phenomena and to evaluate the capabilities of modern computational methods when applied to ion transport. We also strive to draw attention to some underdeveloped areas of ion transport that require further investigation. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium (Na V ) channels are critical in the signal transduction of excitable cells. In this work, we modeled the open conformation for the pore domain of a prokaryotic Na V channel (Na V Rh), and used molecular dynamics simulations to track the translocation of dozens of Na + ions through the channel in the presence of a physiological transmembrane ion concentration gradient and a transmembrane electrical field that was closer to the physiological one than previous studies. Channel conductance was then estimated from simulations on the wide-type and DEKA mutant of Na V Rh. Interestingly, the conductivity predicted from the DEKA mutant agrees well with experimental measurement on eukaryotic Na V 1.4 channel. Moreover, the wide-type and DEKA mutant of Na V Rh exhibited markedly distinct ion permeation patterns, which thus implies the mechanistic difference between prokaryotic and eukaryotic Na V channels. [ABSTRACT FROM AUTHOR]

P2X receptor channels are trimeric ATP-activated ion channels expressed in neuronal and non-neuronal cells that are attractive therapeutic targets for human disorders. Seven subtypes of P2X receptor channels have been identified in mammals that can form both homomeric and heteromeric channels. P2X1-4 and P2X7 receptor channels are cation-selective, whereas P2X5 has been reported to have both cation and anion permeability. P2X receptor channel structures reveal that each subunit is comprised of two transmembrane helices, with both N-and C-termini on the intracellular side of the membrane and a large extracellular domain that contains the ATP binding sites at subunit interfaces. Recent structures of ATP-bound P2X receptors with the activation gate open reveal the unanticipated presence of a cytoplasmic cap over the central ion permeation pathway, leaving lateral fenestrations that may be largely buried within the membrane as potential pathways for ions to permeate the intracellular end of the pore. In the present study, we identify a critical residue within the intracellular lateral fenestrations that is readily accessible to thiol-reactive compounds from both sides of the membrane and where substitutions influence the relative permeability of the channel to cations and anions. Taken together, our results demonstrate that ions can enter or exit the internal pore through lateral fenestrations that play a critical role in determining the ion selectivity of P2X receptor channels., Competing Interests: ST, KH, ML No competing interests declared, KS Senior editor, eLife

Ion transport across cell membranes is essential to cell communication and signaling. Passive ion transport is mediated by ion channels, membrane proteins that create ion conducting pores across cell membrane to allow ion flux down electrochemical gradient. Under physiological conditions, majority of ion channel pores are not constitutively open. Instead, structural region(s) within these pores breaks the continuity of the aqueous ion pathway, thereby serves as activation gate(s) to control ions flow in and out. To achieve spatially and temporally regulated ion flux in cells, many ion channels have evolved sensors to detect various environmental stimuli or the metabolic states of the cell and trigger global conformational changes, thereby dynamically operate the opening and closing of their activation gate. The sensors of ion channels can be broadly categorized as chemical sensors and physical sensors to respond to chemical (such as neural transmitters, nucleotides and ions) and physical (such as voltage, mechanical force and temperature) signals, respectively. With the rapidly growing structural and functional information of different types of ion channels, it is now critical to understand how ion channel sensors dynamically control their gates at molecular and atomic level. The voltage and Ca2+ activated BK channels, a K+ channel with an electrical sensor and multiple chemical sensors, provide a unique model system for us to understand how physical and chemical energy synergistically operate its activation gate.

Purpose: Measurement of skin absorption of ions requires specific experimental protocols regarding the use of pig skin as a model, the viability of excised skin in water medium over 24 h, the presence of endogenous ions, and evaluation of the contributions of facilitated transport through ion channels and ion transporters. Method: Absorption experiments of halide anions F, Cl, Br and I in excised skin were performed in Franz diffusion cells. Experiments were performed on human and porcine skin under various conditions so as to define and validate experimental protocols. Results: The distributions of endogenous ions and the absorption kinetics of halide ions were similar in both porcine and human skin models. Fresh skin kept its viability over 24 h in salt-free water, allowing experiments following OECD guidelines. Permeation increased in the order F < Cl < Br < I for all receptor media and skin samples. Absorption was larger in fresh skin due to the transport through chloride channels or exchangers. Conclusion: Skin absorption experiments of ions in Franz cells rely on working with fresh excised skin (human or porcine) and pure water as receptor fluid. Experiments with chloride blockers or frozen/thawed skin allow discriminating passive diffusion and facilitated transport. [ABSTRACT FROM AUTHOR]

Membrane proteins are embedded in a lipid bilayer and interact with the lipid molecules in subtle ways. This can be studied experimentally by examining the effect of different lipid bilayers on the function of membrane proteins. Understanding the causes of the functional effects of lipids is difficult to dissect experimentally but more amenable to a computational approach. Here we perform molecular dynamics simulations and free energy calculations to study the effect of two lipid types (POPC and NODS) on the conductance of the gramicidin A (gA) channel. A larger energy barrier is found for the K+ potential of mean force in gA embedded in POPC compared to that in NODS, which is consistent with the enhanced experimental conductance of cations in gA embedded in NODS. Further analysis of the contributions to the potential energy of K+ reveals that gA and water molecules in gA make similar contributions in both bilayers but there are significant differences between the two bilayers when the lipid molecules and interfacial waters are considered. It is shown that the stronger dipole moments of the POPC head groups create a thicker layer of interfacial waters with better orientation, which ultimately is responsible for the larger energy barrier in the K+ PMF in POPC. [ABSTRACT FROM AUTHOR]