Atomic structure of a Na+- and K+-conducting channel

Ion selectivity is one of the basic properties that define an ion channel. Most tetrameric cation channels, which include the K+, Ca2+, Na+ and cyclic nucleotide-gated channels, probably share a similar overall architecture in their ion-conduction pore, but the structural details that determine ion selection are different. Although K+ channel selectivity has been well studied from a structural perspective, little is known about the structure of other cation channels. Here we present crystal structures of the NaK channel from Bacillus cereus, a non-selective tetrameric cation channel, in its Na+- and K+-bound states at 2.4 A and 2.8 A resolution, respectively. The NaK channel shares high sequence homology and a similar overall structure with the bacterial KcsA K+ channel, but its selectivity filter adopts a different architecture. Unlike a K+ channel selectivity filter, which contains four equivalent K+-binding sites, the selectivity filter of the NaK channel preserves the two cation-binding sites equivalent to sites 3 and 4 of a K+ channel, whereas the region corresponding to sites 1 and 2 of a K+ channel becomes a vestibule in which ions can diffuse but not bind specifically. Functional analysis using an 86Rb flux assay shows that the NaK channel can conduct both Na+ and K+ ions. We conclude that the sequence of the NaK selectivity filter resembles that of a cyclic nucleotide-gated channel and its structure may represent that of a cyclic nucleotide-gated channel pore.     

Energetic optimization of ion conduction rate by the K+ selectivity filter.

The K+ selectivity filter catalyses the dehydration, transfer and rehydration of a K+ ion in about ten nanoseconds. This physical process is central to the production of electrical signals in biology. Here we show how nearly diffusion-limited rates are achieved, by analysing ion conduction and the corresponding crystallographic ion distribution in the selectivity filter of the KcsA K+ channel. Measurements with K+ and its slightly larger analogue, Rb+, lead us to conclude that the selectivity filter usually contains two K+ ions separated by one water molecule. The two ions move in a concerted fashion between two configurations, K+-water-K+-water (1,3 configuration) and water-K+-water-K+ (2,4 configuration), until a third ion enters, displacing the ion on the opposite side of the queue. For K+, the energy difference between the 1,3 and 2,4 configurations is close to zero, the condition of maximum conduction rate. The energetic balance between these configurations is a clear example of evolutionary optimization of protein function.     

The open pore conformation of potassium channels

Living cells regulate the activity of their ion channels through a process known as gating. To open the pore, protein conformational changes must occur within a channel's membrane-spanning ion pathway. KcsA and MthK, closed and opened K(+) channels, respectively, reveal how such gating transitions occur. Pore-lining 'inner' helices contain a 'gating hinge' that bends by approximately 30 degrees. In a straight conformation four inner helices form a bundle, closing the pore near its intracellular surface. In a bent configuration the inner helices splay open creating a wide (12 A) entryway. Amino-acid sequence conservation suggests a common structural basis for gating in a wide range of K(+) channels, both ligand- and voltage-gated. The open conformation favours high conduction by compressing the membrane field to the selectivity filter, and also permits large organic cations and inactivation peptides to enter the pore from the intracellular solution.     

Energetics of ion conduction through the K+ channel.

K+ channels are transmembrane proteins that are essential for the transmission of nerve impulses. The ability of these proteins to conduct K+ ions at levels near the limit of diffusion is traditionally described in terms of concerted mechanisms in which ion-channel attraction and ion-ion repulsion have compensating effects, as several ions are moving simultaneously in single file through the narrow pore. The efficiency of such a mechanism, however, relies on a delicate energy balance-the strong ion-channel attraction must be perfectly counterbalanced by the electrostatic ion-ion repulsion. To elucidate the mechanism of ion conduction at the atomic level, we performed molecular dynamics free energy simulations on the basis of the X-ray structure of the KcsA K+ channel. Here we find that ion conduction involves transitions between two main states, with two and three K+ ions occupying the selectivity filter, respectively; this process is reminiscent of the 'knock-on' mechanism proposed by Hodgkin and Keynes in 1955. The largest free energy barrier is on the order of 2-3 kcal mol-1, implying that the process of ion conduction is limited by diffusion. Ion-ion repulsion, although essential for rapid conduction, is shown to act only at very short distances. The calculations show also that the rapidly conducting pore is selective.     

Ion permeation mechanism of the potassium channel

Ion-selective channels enable the specific permeation of ions through cell membranes and provide the basis of several important biological functions; for example, electric signalling in the nervous system. Although a large amount of electrophysiological data is available, the molecular mechanisms by which these channels can mediate ion transport remain a significant unsolved problem. With the recently determined crystal structure of the representative K+ channel (KcsA) from Streptomyces lividans, it becomes possible to examine ion conduction pathways on a microscopic level. K+ channels utilize multi-ion conduction mechanisms, and the three-dimensional structure also shows several ions present in the channel. Here we report results from molecular dynamics free energy perturbation calculations that both establish the nature of the multiple ion conduction mechanism and yield the correct ion selectivity of the channel. By evaluating the energetics of all relevant occupancy states of the selectivity filter, we find that the favoured conduction pathway involves transitions only between two main states with a free difference of about 5 kcal mol(-1). Other putative permeation pathways can be excluded because they would involve states that are too high in energy.     

Aspartate 112 is the selectivity filter of the human voltage-gated proton channel

The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage-gated proton channel, H(V)1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes, histamine secretion by basophils, sperm capacitation, and airway pH. The most selective ion channel known, H(V)1 shows no detectable permeability to other ions. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity, or (2) water molecules 'frozen' in a narrow pore conduct protons while excluding other ions. Here we identify aspartate 112 as a crucial component of the selectivity filter of H(V)1. When a neutral amino acid replaced Asp?112, the mutant channel lost proton specificity and became anion-selective or did not conduct. Only the glutamate mutant remained proton-specific. Mutation of the nearby Asp?185 did not impair proton selectivity, indicating that Asp?112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp?112, the mutant channel was still anion-permeable. Evidently, the proton specificity of H(V)1 requires an acidic group at the selectivity filter.     

KcsA一种新型的K离子通道

Declan A Doyle et al于1998年利用X射线结晶分析在Streptomyces lividans(变铅青链霉菌)中发现的KcsA(K^ conduction and selectivity architecture)是一种新型的K^ 通道。它由四个亚基组成，每个亚基含有两个α—螺旋，在KcsA的中央有一个选择性滤膜，对K^ 具有特殊的通透性。本文仅对KcsA的结构及其对K^ 选择性介导的作用机制进行综述。     

Control of ion selectivity in potassium channels by electrostatic and dynamic properties of carbonyl ligands

Potassium channels are essential for maintaining a normal ionic balance across cell membranes. Central to this function is the ability of such channels to support transmembrane ion conduction at nearly diffusion-limited rates while discriminating for K+ over Na+ by more than a thousand-fold. This selectivity arises because the transfer of the K+ ion into the channel pore is energetically favoured, a feature commonly attributed to a structurally precise fit between the K+ ion and carbonyl groups lining the rigid and narrow pore. But proteins are relatively flexible structures that undergo rapid thermal atomic fluctuations larger than the small difference in ionic radius between K+ and Na+. Here we present molecular dynamics simulations for the potassium channel KcsA, which show that the carbonyl groups coordinating the ion in the narrow pore are indeed very dynamic ('liquid-like') and that their intrinsic electrostatic properties control ion selectivity. This finding highlights the importance of the classical concept of field strength. Selectivity for K+ is seen to emerge as a robust feature of a flexible fluctuating pore lined by carbonyl groups.     

The crystal structure of a voltage-gated sodium channel

Voltage-gated sodium (Na(V)) channels initiate electrical signalling in excitable cells and are the molecular targets for drugs and disease mutations, but the structural basis for their voltage-dependent activation, ion selectivity and drug block is unknown. Here we report the crystal structure of a voltage-gated Na(+) channel from Arcobacter butzleri (NavAb) captured in a closed-pore conformation with four activated voltage sensors at 2.7?? resolution. The arginine gating charges make multiple hydrophilic interactions within the voltage sensor, including unanticipated hydrogen bonds to the protein backbone. Comparisons to previous open-pore potassium channel structures indicate that the voltage-sensor domains and the S4-S5 linkers dilate the central pore by pivoting together around a hinge at the base of the pore module. The NavAb selectivity filter is short, ～4.6?? wide, and water filled, with four acidic side chains surrounding the narrowest part of the ion conduction pathway. This unique structure presents a high-field-strength anionic coordination site, which confers Na(+) selectivity through partial dehydration via direct interaction with glutamate side chains. Fenestrations in the sides of the pore module are unexpectedly penetrated by fatty acyl chains that extend into the central cavity, and these portals are large enough for the entry of small, hydrophobic pore-blocking drugs. This structure provides the template for understanding electrical signalling in excitable cells and the actions of drugs used for pain, epilepsy and cardiac arrhythmia at the atomic level.     

无机盐作用下伊利石水化特性的分子模拟

伊蒙混层水化导致井壁失稳的现象相当突出，蒙脱石的水化研究较多。为研究伊利石在无机盐作用下的水化机理及膨胀特性，通过分子模拟技术建立了1M-tv和1M-cv两种在油气储层中较常见的伊利石晶体模型，并进一步通过分子动力学方法研究了伊利石层间粒子的微观分布和进入层间的无机盐阳离子的水化参数。结果表明，伊利石层间距随吸附水分子数的增大而增大，当水分子数为20时达到饱和状态； 1M-tv构型比1M-cv构型更容易发生水化膨胀； K⁺和Ca²⁺分别是一价阳离子和二价阳离子中水化数和水化半径最小的，且K⁺可以嵌入四面体片层的硅氧六元环中而难以发生离子交换，可有效阻止水分子进入伊利石晶体结构内部；实验结果很好地验证了KCl和CaCl₂对伊利石水化膨胀的抑制性。该结果对于页岩的防塌机理研究和钻井液水化抑制剂的研究具有一定的理论指导意义。     

Calcium channel characteristics conferred on the sodium channel by single mutations.

The sodium channel, one of the family of structurally homologous voltage-gated ion channels, differs from other members, such as the calcium and the potassium channels, in its high selectivity for Na+. This selectivity presumably reflects a distinct structure of its ion-conducting pore. We have recently identified two clusters of predominantly negatively charged amino-acid residues, located at equivalent positions in the four internal repeats of the sodium channel as the main determinants of sensitivity to the blockers tetrodotoxin and saxitoxin. All site-directed mutations reducing net negative charge at these positions also caused a marked decrease in single-channel conductance. Thus these two amino-acid clusters probably form part of the extracellular mouth and/or the pore wall of the sodium channel. We report here the effects on ion selectivity of replacing lysine at position 1,422 in repeat III and/or alanine at position 1,714 in repeat IV of rat sodium channel II (ref. 3), each located in one of the two clusters, by glutamic acid, which occurs at the equivalent positions in calcium channels. These amino-acid substitutions, unlike other substitutions in the adjacent regions, alter ion-selection properties of the sodium channel to resemble those of calcium channels. This result indicates that lysine 1,422 and alanine 1,714 are critical in determining the ion selectivity of the sodium channel, suggesting that these residues constitute part of the selectivity filter of the channel.     

Alteration of ionic selectivity of a K+ channel by mutation of the H5 region

The high ionic selectivity of K+ channels is a unifying feature of this diverse class of membrane proteins. Though K+ channels differ widely in regulation and kinetics, physiological studies have suggested a common structure: a single file pore containing multiple ion-binding sites and having broader vestibules at both ends. We have used site-directed mutagenesis and single-channel recordings to identify a molecular region that influences ionic selectivity in a cloned A-type K+ channel from Drosophila. Single amino-acid substitutions in H5, the fifth hydrophobic region, enhanced the passage of NH4+ and Rb+, ions with diameters larger than K+, without compromising the ability of the channel to exclude the smaller cation, Na+. The mutations that substantially altered selectivity had little effect on the gating properties of the channel. We conclude that the H5 region is likely to line the pore of the K+ channel.     

Interaction between liquid water and hydroxide revealed by core-hole de-excitation

The hydroxide ion plays an important role in many chemical and biochemical processes in aqueous solution. But our molecular-level understanding of its unusual and fast transport in water, and of the solvation patterns that allow fast transport, is far from complete. One proposal seeks to explain the properties and behaviour of the hydroxide ion by essentially regarding it as a water molecule that is missing a proton, and by inferring transport mechanisms and hydration structures from those of the excess proton. A competing proposal invokes instead unique and interchanging hydroxide hydration complexes, particularly the hypercoordinated OH(-)(H(2)O)(4) species and tri-coordinated OH(-)(H(2)O)(3) that can form a transient hydrogen bond between the H atom of the OH(-) and a neighbouring water molecule. Here we report measurements of core-level photoelectron emission and intermolecular Coulombic decay for an aqueous hydroxide solution, which show that the hydrated hydroxide ion is capable of transiently donating a hydrogen bond to surrounding water molecules. In agreement with recent experimental studies of hydroxide solutions, our finding thus supports the notion that the hydration structure of the hydroxide ion cannot be inferred from that of the hydrated excess proton.     

Crystal structure of a potassium ion transporter, TrkH

The TrkH/TrkG/KtrB proteins mediate K(+) uptake in bacteria and probably evolved from simple K(+) channels by multiple gene duplications or fusions. Here we present the crystal structure of a TrkH from Vibrio parahaemolyticus. TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to those of K(+) channels but significantly shorter, is lined by backbone and side-chain oxygen atoms. Functional studies showed that TrkH is selective for permeation of K(+) and Rb(+) over smaller ions such as Na(+) or Li(+). Immediately intracellular to the selectivity filter are an intramembrane loop and an arginine residue, both highly conserved, which constrict the permeation pathway. Substituting the arginine with an alanine significantly increases the rate of K(+) flux. These results reveal the molecular basis of K(+) selectivity and suggest a novel gating mechanism for this large and important family of membrane transport proteins.     

Structural basis of water-specific transport through the AQP1 water channel.

Water channels facilitate the rapid transport of water across cell membranes in response to osmotic gradients. These channels are believed to be involved in many physiological processes that include renal water conservation, neuro-homeostasis, digestion, regulation of body temperature and reproduction. Members of the water channel superfamily have been found in a range of cell types from bacteria to human. In mammals, there are currently 10 families of water channels, referred to as aquaporins (AQP): AQP0-AQP9. Here we report the structure of the aquaporin 1 (AQP1) water channel to 2.2 A resolution. The channel consists of three topological elements, an extracellular and a cytoplasmic vestibule connected by an extended narrow pore or selectivity filter. Within the selectivity filter, four bound waters are localized along three hydrophilic nodes, which punctuate an otherwise extremely hydrophobic pore segment. This unusual combination of a long hydrophobic pore and a minimal number of solute binding sites facilitates rapid water transport. Residues of the constriction region, in particular histidine 182, which is conserved among all known water-specific channels, are critical in establishing water specificity. Our analysis of the AQP1 pore also indicates that the transport of protons through this channel is highly energetically unfavourable.     

Ion channel of acetylcholine receptor reconstructed from images of postsynaptic membranes

The nicotinic acetylcholine receptor belongs to a class of molecules that respond transiently to chemical stimuli by opening a water-filled channel through the cell membrane for cations to diffuse. This channel lies along the central axis delineated by a ring of five homologous, membrane-spanning subunits and thus has properties, such as conductance and ion selectivity, which depend on the profile created by the encircling subunits. Insight has been gained recently about the amino-acid residues implicated directly in the ion transport, and some information about the subunit configuration around the channel has come from electron microscopy studies of postsynaptic membranes crystallized in the form of flattened tubular vesicles. The resolution along the axis of the channel has, however, been limited by the restricted range of views obtainable. Here we report the structure of the channel at 17 A resolution, determined by three-dimensional image reconstruction from tubular vesicles having receptors organized in helical arrays across their surfaces. The helical symmetry is preserved by suspending the tubes in thin films of ice, and the receptors in such tubes can be seen from all angles, allowing the channel to be revealed clearly in relation to the lipid bilayer and the peripheral protein for the first time.     

Homologue structure of the SLAC1 anion channel for closing stomata in leaves

The plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating the exchange of water vapour and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. Here we determine the crystal structure of a bacterial homologue (Haemophilus influenzae) of SLAC1 at 1.20 ? resolution, and use structure-inspired mutagenesis to analyse the conductance properties of SLAC1 channels. SLAC1 is a symmetrical trimer composed from quasi-symmetrical subunits, each having ten transmembrane helices arranged from helical hairpin pairs to form a central five-helix transmembrane pore that is gated by an extremely conserved phenylalanine residue. Conformational features indicate a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics indicate that selectivity among different anions is largely a function of the energetic cost of ion dehydration.     

黑云母水化机理及对注水开发影响

从黑云母的化学结构和结晶形态出发,本质上分析了黑云母的水化机理,黑云母水化的实质是层间域中K+被其他大分子或离子替换,是一个释放K+的过程。水化过程可分为解理缝裂开、边缘断裂分散为特征的边缘水化阶段和解理缝消失、颗粒呈鳞片状为特征的完全水化阶段。黑云母一旦在储层中水化,晶体结构膨胀,部分边缘颗粒逐渐脱落,随水动力分散运移,堵塞孔喉,易造成储层损害。含黑云母储层在注水开发中应该提高K+浓度,防止云母水化产物产生的水敏和速敏损害,以保护储集层。     

Correlation between surface charge and hydration on mineral surfaces in aqueous solutions: A critical review

Surface charges and hydration are predominant properties of colloidal particles that govern colloidal stability in aqueous suspensions. These properties usually coexist and interact with each other. The correlation between the surface charge and hydration of minerals is summarized on the basis of innovative experimental, theoretical, and molecular dynamics simulation studies. The factors affecting the adsorption behavior of ions and water molecules, such as ion concentration, ion hydration radius and valence, and surface properties, are discussed.For example, the hydration and adsorption states completely differ between monovalent and divalent ions. For ions of the same valence, the effect of surface charge on the hydration force follows the Hofmeister adsorption series. Electrolyte concentration exerts a significant effect on the hydration force at high ion concentrations. Meanwhile, the ion correlations in high-concentration electrolyte systems become long range.The interfacial water structure largely depends on surface chemistry. The hydration layer between different surfaces shows large qualitative differences.     

Single stretch-activated ion channels in vascular endothelial cells as mechanotransducers?

Endothelial cells line the inner surface of blood vessels and act as the main barrier to the passage of cells and large molecules from the blood stream to the tissues. Recent interest in the part played by the endothelium in regulating vascular tone has focused on the synthesis and secretion of prostacyclin and an endothelium-derived relaxing factor. Endothelial cells respond to blood-borne agonist, but how the endothelium senses and responds to mechanical forces generated by the flow of blood under pressure is not known. Here we report patch-clamp recordings of ion channel activity from cell-attached membrane patches on aortic endothelial cells. In most of the patches examined, we observed unitary inward currents associated with the opening of a cation-selective channel (approximately 40 pS in standard saline). The channel is permeable to Ca2+ and its opening frequency increases when the membrane is stretched by applying suction through the patch electrode. The presence of mechanotransducing ion channels in endothelial cells may help explain how the endothelium mediates vascular responses to haemodynamic stresses.