Exchange of conduction pathways between two related K+ channels

The structure of the ion conduction pathway or pore of voltage-gated ion channels is unknown, although the linker between the membrane spanning segments S5 and S6 has been suggested to form part of the pore in potassium channels. To test whether this region controls potassium channel conduction, a 21-amino acid segment of the S5-S6 linker was transplanted from the voltage-activated potassium channel NGK2 to another potassium channel DRK1, which has very different pore properties. In the resulting chimeric channel, the single channel conductance and blockade by external and internal tetraethylammonium (TEA) ion were characteristic of the donor NGK2 channel. Thus, this 21-amino acid segment controls the essential biophysical properties of the pore and may form the conduction pathway of these potassium channels.     

Mutations affecting TEA blockade and ion permeation in voltage-activated K+ channels

Voltage-dependent ion channels are responsible for electrical signaling in neurons and other cells. The main classes of voltage-dependent channels (sodium-, calcium-, and potassium-selective channels) have closely related molecular structures. For one member of this superfamily, the transiently voltage-activated Shaker H4 potassium channel, specific amino acid residues have now been identified that affect channel blockade by the small ion tetraethylammonium, as well as the conduction of ions through the pore. Furthermore, variation at one of these amino acid positions among naturally occurring potassium channels may account for most of their differences in sensitivity to tetraethylammonium.     

商陆PaHAK1与拟南芥HAK/KUP/KT家族基因的比较分析

为探明商陆高亲和性K+转运体基因(Pa HAK1)的结构、功能及商陆耐低钾的原因,分析比较了商陆Pa HAK1和拟南芥HAK/KUP/KT家族基因中15个基因编码的高亲和性钾离子转运体氨基酸序列、蛋白质结构及其理化性质。结果表明:HAK基因家族编码蛋白均定位于细胞膜上,含有多个跨膜结构且跨膜结构域是蛋白质的保守结构域;Pa HAK1与At KUP3的跨膜结构十分相似,低钾胁迫下Pa HAK1和At KUP3的高表达与其高亲和钾转运吸收功能密切相关。系统进化树分析结果表明,Pa HAK1与拟南芥HAK基因家族中At KUP8亲缘关系最近,其氨基酸序列相似度为76.98%,推测Pa HAK1还可能通过维持细胞内高水平钾量在水分胁迫下发挥重要的调节作用。     

Stretch-inactivated ion channels coexist with stretch-activated ion channels

Stretch-activated ion channels of animal, plant, bacterial, and fungal cells are implicated in mechanotransduction and osmoregulation. A new class of channel has now been described that is stretch-inactivated. These channels occur in neurons, where they coexist with stretch-activated channels. Both channels are potassium selective. The differing stretch sensitivities of the two channels minimize potassium conductance over an intermediate range of tension, with the consequence that, over this same range, voltage-gated calcium channels are most readily opened. Thus, by setting the relation between membrane tension and transmembrane calcium fluxes, stretch-sensitive potassium channels may participate in the control of calcium-dependent motility in differentiating, regenerating, or migrating neurons.     

The gramicidin pore: crystal structure of a cesium complex

Gramicidin, a linear polypeptide composed of hydrophobic amino acids with alternating L- and D- configurations, forms transmembrane ion channels. The crystal structure of a gramicidin-cesium complex has been determined at 2.0 angstrom resolution. In this structure, gramicidin forms a 26 angstrom long tube comprised of two polypeptide chains arranged as antiparallel beta strands that are wrapped into a left-handed helical coil with 6.4 residues per turn. The polypeptide backbone forms the interior of the hydrophilic, solvent-filled pore and the side chains form a hydrophobic and relatively regular surface on the outside of the pore. This example of a crystal structure of a solvent-filled ion pore provides a basis for understanding the physical nature of ion translocation.     

Gating the selectivity filter in ClC chloride channels

ClC channels conduct chloride (Cl-) ions across cell membranes and thereby govern the electrical activity of muscle cells and certain neurons, the transport of fluid and electrolytes across epithelia, and the acidification of intracellular vesicles. The structural basis of ClC channel gating was studied. Crystal structures of wild-type and mutant Escherichia coli ClC channels bound to a monoclonal Fab fragment reveal three Cl- binding sites within the 15-angstrom neck of an hourglass-shaped pore. The Cl- binding site nearest the extracellular solution can be occupied either by a Cl- ion or by a glutamate carboxyl group. Mutations of this glutamate residue in Torpedo ray ClC channels alter gating in electrophysiological assays. These findings reveal a form of gating in which the glutamate carboxyl group closes the pore by mimicking a Cl- ion.     

植物根细胞离子通道研究进展

根细胞膜上存在各种离子通道.电生理学的研究表明,根细胞离子通道对于矿质吸收、转运及植物耐盐具有重要作用.该文概述了根细胞K+通道、阴离子通道和各种非选择性阳离子通道的最新研究进展,并对近期有关离子通道和植物耐盐性关系的研究进行了总结.K+通道存在于大多数的植物细胞中,其对K+的选择性远高于其他阳离子,K+通道的存在对于营养元素的吸收,尤其是K+的低亲和性吸收具有重要的意义,同时也为其他离子的出入维持了一个较为稳定的膜电势.阴离子通道激活所引起的质膜去极化可以激发非选择性的阳离子流,在盐胁迫下,可通透Cl的阴离子通道的开放是植物对胞内Cl的一种重要调控机制.由于非选择性的阳离子通道(Non-selective cation channels,NSCCs)的多样性及其对一价阳离子的低选择性,近年来NSCCs的研究受到广泛关注.NSCCs被认为参与了植物多种生理过程,包括营养元素的吸收、膨压控制、胞间转运、信号转导以及毒害离子的吸收,尤其是Na+的吸收.      

Changes in sodium channel gating produced by point mutations in a cytoplasmic linker

Voltage-gated sodium channels are transmembrane proteins of approximately 2000 amino acids and consist of four homologous domains (I through IV). In current topographical models, domains III and IV are linked by a highly conserved cytoplasmic sequence of amino acids. Disruptions of the III-IV linker by cleavage or antibody binding slow inactivation, the depolarization-induced closed state characteristic of sodium channels. This linker might be the positively charged "ball" that is thought to cause inactivation by occluding the open channel. Therefore, groups of two or three contiguous lysines were neutralized or a glutamate was substituted for an arginine in the III-IV linker of type III rat brain sodium channels. In all cases, inactivation occurred more rapidly rather than more slowly, contrary to predictions. Furthermore, activation was delayed in the arginine to glutamate mutation. Hence, the III-IV linker does not simply act as a charged blocker of the channel but instead influences all aspects of sodium channel gating.     

Voltage sensor of Kv1.2: structural basis of electromechanical coupling

Voltage-dependent ion channels contain voltage sensors that allow them to switch between nonconductive and conductive states over the narrow range of a few hundredths of a volt. We investigated the mechanism by which these channels sense cell membrane voltage by determining the x-ray crystal structure of a mammalian Shaker family potassium ion (K+) channel. The voltage-dependent K+ channel Kv1.2 grew three-dimensional crystals, with an internal arrangement that left the voltage sensors in an apparently native conformation, allowing us to reach three important conclusions. First, the voltage sensors are essentially independent domains inside the membrane. Second, they perform mechanical work on the pore through the S4-S5 linker helices, which are positioned to constrict or dilate the S6 inner helices of the pore. Third, in the open conformation, two of the four conserved Arg residues on S4 are on a lipid-facing surface and two are buried in the voltage sensor. The structure offers a simple picture of how membrane voltage influences the open probability of the channel.     

Occult Drosophila calcium channels and twinning of calcium and voltage-activated potassium channels

In the membrane of the flight muscle cells of developing Drosophila a large calcium-sensitive potassium current, IKc, was found. It was present before the development of voltage-activated potassium channels and seems to be the first potassium current to develop in the membrane. Also present in these early cells were large numbers of occult (hidden) calcium channels, which remained inactive until the end of pupal development. These inactive calcium channels could be made to function by injecting adenosine triphosphate or ethyleneglycol tetraacetic acid into the early cells. IKc has kinetic properties resembling the later developing voltage-sensitive current IKv, and is distinct from the fast, transient calcium-dependent outward current IAc, which appears much later in development. IAc closely resembles the voltage-sensitive current IAv, also present in these cells. Thus, both of the voltage-sensitive potassium channel types, IAv and IKv, have similar calcium-sensitive counterparts, IAc and IKc, that are present in the same cells.     

A component of calcium-activated potassium channels encoded by the Drosophila slo locus.

Calcium-activated potassium channels mediate many biologically important functions in electrically excitable cells. Despite recent progress in the molecular analysis of voltage-activated K+ channels, Ca(2+)-activated K+ channels have not been similarly characterized. The Drosophila slowpoke (slo) locus, mutations of which specifically abolish a Ca(2+)-activated K+ current in muscles and neurons, provides an opportunity for molecular characterization of these channels. Genomic and complementary DNA clones from the slo locus were isolated and sequenced. The polypeptide predicted by slo is similar to voltage-activated K+ channel polypeptides in discrete domains known to be essential for function. Thus, these results indicate that slo encodes a structural component of Ca(2+)-activated K+ channels.     

Mechanism of voltage gating in potassium channels

The mechanism of ion channel voltage gating-how channels open and close in response to voltage changes-has been debated since Hodgkin and Huxley's seminal discovery that the crux of nerve conduction is ion flow across cellular membranes. Using all-atom molecular dynamics simulations, we show how a voltage-gated potassium channel (KV) switches between activated and deactivated states. On deactivation, pore hydrophobic collapse rapidly halts ion flow. Subsequent voltage-sensing domain (VSD) relaxation, including inward, 15-angstrom S4-helix motion, completes the transition. On activation, outward S4 motion tightens the VSD-pore linker, perturbing linker-S6-helix packing. Fluctuations allow water, then potassium ions, to reenter the pore; linker-S6 repacking stabilizes the open pore. We propose a mechanistic model for the sodium/potassium/calcium voltage-gated ion channel superfamily that reconciles apparently conflicting experimental data.     

Steady-state coupling of ion-channel conformations to a transmembrane ion gradient

Under stationary conditions, opening and closing of single Torpedo electroplax chloride channels show that the number of transitions per unit time between inactivated and conducting states are unequal in opposite directions. This asymmetry, which increases with transmembrane electrochemical gradient for the chloride ion, violates the principle of microscopic reversibility and thus demonstrates that the channel-gating process is not at thermodynamic equilibrium. The results imply that the channel's conformational states are coupled to the transmembrane electrochemical gradient of the chloride ion.     

Calcium-sensitive inactivation in the gating of single calcium channels

Voltage-activated calcium channels open and close, or gate, according to molecular transition rates that are regulated by transmembrane voltage and neurotransmitters. Here evidence for the control of gating by calcium was found in electrophysiological records of single, L-type calcium channels in heart cells. Conditional open probability analysis revealed that calcium entry during the opening of a single channel produces alterations in gating transition rates that evolve over the course of hundreds of milliseconds. Such alteration of calcium-channel gating by entry of a favored permeant ion provides a mechanism for the short-term modulation of single-ion channels.     

Pedhn基因的克隆及表达分析

为深入研究胡杨的抗盐分子机制,并获得与之相关的抗逆基因,采用SMART技术构建了胡杨盐胁迫处理cDNA文库。经随机测序,得到一个编号为SSL061的EST序列,与欧美杨的脱水素基因有较高相似性,通过PCR快速扩增文库的方法获得了胡杨脱水素基因（Pedhn）。分析表明,该cDNA的长度为813 bp,拥有681 bp 的开放读码框,共编码227个氨基酸。该序列包括两个重复的富含赖氨酸的K片段和由7个连续的丝氨酸残基组成的S片段,但是缺乏Y片段。RT-PCR结果表明,胡杨在未受胁迫的情况下脱水素基因的转录水平较低,而随着盐胁迫浓度的升高,脱水素基因的转录水平总体呈上升趋势。      

The structure of an open form of an E. coli mechanosensitive channel at 3.45 A resolution

How ion channels are gated to regulate ion flux in and out of cells is the subject of intense interest. The Escherichia coli mechanosensitive channel, MscS, opens to allow rapid ion efflux, relieving the turgor pressure that would otherwise destroy the cell. We present a 3.45 angstrom-resolution structure for the MscS channel in an open conformation. This structure has a pore diameter of approximately 13 angstroms created by substantial rotational rearrangement of the three transmembrane helices. The structure suggests a molecular mechanism that underlies MscS gating and its decay of conductivity during prolonged activation. Support for this mechanism is provided by single-channel analysis of mutants with altered gating characteristics.     

Cloning of a membrane protein that induces a slow voltage-gated potassium current

A rat kidney messenger RNA that induces a slowly activating, voltage-dependent potassium current on its expression in Xenopus oocytes was identified by combining molecular cloning with an electrophysiological assay. The cloned complementary DNA encodes a novel membrane protein that consists of 130 amino acids with a single putative transmembrane domain. This protein differs from the known ion channel proteins but is involved in the induction of selective permeation of potassium ions by membrane depolarization.     

Glycolysis preferentially inhibits ATP-sensitive K+ channels in isolated guinea pig cardiac myocytes

In heart, glycolysis may be a preferential source of adenosine triphosphate (ATP) for membrane functions. In this study the patch-clamp technique was used to study potassium channels sensitive to intracellular ATP levels in permeabilized ventricular myocytes. Activation of these K+ channels has been implicated in marked cellular K+ loss leading to electrophysiological abnormalities and arrhythmias during myocardial ischemia. The results showed that glycolysis was more effective than oxidative phosphorylation in preventing ATP-sensitive K+ channels from opening. Experiments in excised inside-out patches suggested that key glycolytic enzymes located in the membrane or adjacent cytoskeleton near the channels may account for their preference for glycolytic ATP.     

Ca2+ entry through plasma membrane IP3 receptors

Inositol 1,4,5-trisphosphate receptors (IP3Rs) release calcium ions, Ca2+, from intracellular stores, but their roles in mediating Ca2+ entry are unclear. IP3 stimulated opening of very few (1.9 +/- 0.2 per cell) Ca2+-permeable channels in whole-cell patch-clamp recording of DT40 chicken or mouse B cells. Activation of the B cell receptor (BCR) in perforated-patch recordings evoked the same response. IP3 failed to stimulate intracellular or plasma membrane (PM) channels in cells lacking IP3R. Expression of IP3R restored both responses. Mutations within the pore affected the conductances of IP3-activated PM and intracellular channels similarly. An impermeant pore mutant abolished BCR-evoked Ca2+ signals, and PM IP3Rs were undetectable. After introduction of an alpha-bungarotoxin binding site near the pore, PM IP3Rs were modulated by extracellular alpha-bungarotoxin. IP(3)Rs are unusual among endoplasmic reticulum proteins in being also functionally expressed at the PM, where very few IP3Rs contribute substantially to the Ca2+ entry evoked by the BCR.     

Crystal structure of a mammalian voltage-dependent Shaker family K+ channel

Voltage-dependent potassium ion (K+) channels (Kv channels) conduct K+ ions across the cell membrane in response to changes in the membrane voltage, thereby regulating neuronal excitability by modulating the shape and frequency of action potentials. Here we report the crystal structure, at a resolution of 2.9 angstroms, of a mammalian Kv channel, Kv1.2, which is a member of the Shaker K+ channel family. This structure is in complex with an oxido-reductase beta subunit of the kind that can regulate mammalian Kv channels in their native cell environment. The activation gate of the pore is open. Large side portals communicate between the pore and the cytoplasm. Electrostatic properties of the side portals and positions of the T1 domain and beta subunit are consistent with electrophysiological studies of inactivation gating and with the possibility of K+ channel regulation by the beta subunit.