ATP-sensitive K+ channel in the mitochondrial inner membrane.

Mitochondria take up and extrude various inorganic and organic ions, as well as larger substances such as proteins. The technique of patch clamping should provide real-time information on such transport and on energy transduction in oxidative phosphorylation. It has been applied to detect microscopic currents from mitochondrial membranes and conductances of ion channels in the 5-1,000 pS range in the outer and inner membranes. These pores are not, however, selective for particular ions. Here we use fused giant mitoplasts prepared from rat liver mitochondria to identify a small conductance channel highly selective for K+ in the inner mitochondrial membrane. This channel can be reversibly inactivated by ATP applied to the matrix side under inside-out patch configuration; it is also inhibited by 4-aminopyridine and by glybenclamide. The slope conductance of the unitary currents measured at negative membrane potentials was 9.7 +/- 1.0 pS (mean +/- s.d., n = 6) when the pipette solution contained 100 mM K+ and the bathing solution 33.3 mM K+. Our results indicate that mitochondria depolarize by generating a K+ conductance when ATP in the matrix is deficient.     

The mitochondrial calcium uniporter is a highly selective ion channel

During intracellular Ca2+ signalling mitochondria accumulate significant amounts of Ca2+ from the cytosol. Mitochondrial Ca2+ uptake controls the rate of energy production, shapes the amplitude and spatio-temporal patterns of intracellular Ca2+ signals, and is instrumental to cell death. This Ca2+ uptake is undertaken by the mitochondrial Ca2+ uniporter (MCU) located in the organelle's inner membrane. The uniporter passes Ca2+ down the electrochemical gradient maintained across this membrane without direct coupling to ATP hydrolysis or transport of other ions. Carriers are characterized by turnover numbers that are typically 1,000-fold lower than ion channels, and until now it has been unclear whether the MCU is a carrier or a channel. By patch-clamping the inner mitochondrial membrane, we identified a previously unknown Ca2+-selective ion channel sensitive to inhibitors of mitochondrial Ca2+ uptake. Our data indicate that this unique channel binds Ca2+ with extremely high affinity (dissociation constant < or =2 nM), enabling high Ca2+ selectivity despite relatively low cytoplasmic Ca2+ concentrations. The channel is inwardly rectifying, making it especially effective for Ca2+ uptake into energized mitochondria. Thus, we conclude that the properties of the current mediated by this novel channel are those of the MCU.     

X-ray structure of a voltage-dependent K+ channel

Voltage-dependent K+ channels are members of the family of voltage-dependent cation (K+, Na+ and Ca2+) channels that open and allow ion conduction in response to changes in cell membrane voltage. This form of gating underlies the generation of nerve and muscle action potentials, among other processes. Here we present the structure of KvAP, a voltage-dependent K+ channel from Aeropyrum pernix. We have determined a crystal structure of the full-length channel at a resolution of 3.2 A, and of the isolated voltage-sensor domain at 1.9 A, both in complex with monoclonal Fab fragments. The channel contains a central ion-conduction pore surrounded by voltage sensors, which form what we call 'voltage-sensor paddles'-hydrophobic, cationic, helix-turn-helix structures on the channel's outer perimeter. Flexible hinges suggest that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.     

Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment

Voltage-dependent K+ (Kv) channels repolarize the action potential in neurons and muscle. This type of channel is gated directly by membrane voltage through protein domains known as voltage sensors, which are molecular voltmeters that read the membrane voltage and regulate the pore. Here we describe the structure of a chimaeric voltage-dependent K+ channel, which we call the 'paddle-chimaera channel', in which the voltage-sensor paddle has been transferred from Kv2.1 to Kv1.2. Crystallized in complex with lipids, the complete structure at 2.4 ?ngstr?m resolution reveals the pore and voltage sensors embedded in a membrane-like arrangement of lipid molecules. The detailed structure, which can be compared directly to a large body of functional data, explains charge stabilization within the membrane and suggests a mechanism for voltage-sensor movements and pore gating.     

Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death

The t(14; 18) chromosomal translocation of human follicular B-cell lymphoma juxtaposes the bcl-2 gene with the immunoglobulin heavy chain locus. The bcl-2 immunoglobulin fusion gene is markedly deregulated resulting in inappropriately elevated levels of bcl-2 RNA and protein. Transgenic mice bearing a bcl-2 immunoglobulin minigene demonstrate a polyclonal expansion of resting yet responsive IgM-IgD B cells which display prolonged cell survival but no increase in cell cycling. Moreover, deregulated bcl-2 extends the survival of certain haematopoietic cell lines following growth-factor deprivation. By using immunolocalization studies we now demonstrate that Bcl-2 is an integral inner mitochondrial membrane protein of relative molecular mass 25,000 (25k). Overexpression of Bcl-2 blocks the apoptotic death of a pro-B-lymphocyte cell line. Thus, Bcl-2 is unique among proto-oncogenes, being localized to mitochondria and interfering with programmed cell death independent of promoting cell division.     

The principle of gating charge movement in a voltage-dependent K+ channel

The steep dependence of channel opening on membrane voltage allows voltage-dependent K+ channels to turn on almost like a switch. Opening is driven by the movement of gating charges that originate from arginine residues on helical S4 segments of the protein. Each S4 segment forms half of a 'voltage-sensor paddle' on the channel's outer perimeter. Here we show that the voltage-sensor paddles are positioned inside the membrane, near the intracellular surface, when the channel is closed, and that the paddles move a large distance across the membrane from inside to outside when the channel opens. KvAP channels were reconstituted into planar lipid membranes and studied using monoclonal Fab fragments, a voltage-sensor toxin, and avidin binding to tethered biotin. Our findings lead us to conclude that the voltage-sensor paddles operate somewhat like hydrophobic cations attached to levers, enabling the membrane electric field to open and close the pore.     

Functional analysis of an archaebacterial voltage-dependent K+ channel

All living organisms use ion channels to regulate the transport of ions across cellular membranes. Certain ion channels are classed as voltage-dependent because they have a voltage-sensing structure that induces their pores to open in response to changes in the cell membrane voltage. Until recently, the voltage-dependent K+, Ca2+ and Na+ channels were regarded as a unique development of eukaryotic cells, adapted to accomplish specialized electrical signalling, as exemplified in neurons. Here we present the functional characterization of a voltage-dependent K+ (K(V)) channel from a hyperthermophilic archaebacterium from an oceanic thermal vent. This channel possesses all the functional attributes of classical neuronal K(V) channels. The conservation of function reflects structural conservation in the voltage sensor as revealed by specific, high-affinity interactions with tarantula venom toxins, which evolved to inhibit eukaryotic K(V) channels.     

Polymer inaccessible volume changes during opening and closing of a voltage-dependent ionic channel

Osmotic stress can be used to estimate the internal volume change during the opening and closing of a voltage gated ionic channel. Mitochondrial voltage-dependent anion channels, from rat liver and from Neurospora, reconstituted into planar lipid bilayers show a change of 2 to 4 X 10(4) A3 in internal volume, a large change inconsistent with a blocking or local gating model but supporting models with major closure of the channel space.     

X-ray structure of a prokaryotic pentameric ligand-gated ion channel

Pentameric ligand-gated ion channels (pLGICs) are key players in the early events of electrical signal transduction at chemical synapses. The family codes for a structurally conserved scaffold of channel proteins that open in response to the binding of neurotransmitter molecules. All proteins share a pentameric organization of identical or related subunits that consist of an extracellular ligand-binding domain followed by a transmembrane channel domain. The nicotinic acetylcholine receptor (nAChR) is the most thoroughly studied member of the pLGIC family (for recent reviews see refs 1-3). Two sources of structural information provided an architectural framework for the family. The structure of the soluble acetylcholine-binding protein (AChBP) defined the organization of the extracellular domain and revealed the chemical basis of ligand interaction. Electron microscopy studies of the nAChR from Torpedo electric ray have yielded a picture of the full-length protein and have recently led to the interpretation of an electron density map at 4.0 A resolution. Despite the wealth of experimental information, high-resolution structures of any family member have so far not been available. Until recently, the pLGICs were believed to be only expressed in multicellular eukaryotic organisms. The abundance of prokaryotic genome sequences, however, allowed the identification of several homologous proteins in bacterial sources. Here we present the X-ray structure of a prokaryotic pLGIC from the bacterium Erwinia chrysanthemi (ELIC) at 3.3 A resolution. Our study reveals the first structure of a pLGIC at high resolution and provides an important model system for the investigation of the general mechanisms of ion permeation and gating within the family.     

Identification and characterization of a bacterial hydrosulphide ion channel

The hydrosulphide ion (HS(-)) and its undissociated form, hydrogen sulphide (H(2)S), which are believed to have been critical to the origin of life on Earth, remain important in physiology and cellular signalling. As a major metabolite in anaerobic bacterial growth, hydrogen sulphide is a product of both assimilatory and dissimilatory sulphate reduction. These pathways can reduce various oxidized sulphur compounds including sulphate, sulphite and thiosulphate. The dissimilatory sulphate reduction pathway uses this molecule as the terminal electron acceptor for anaerobic respiration, in which process it produces excess amounts of H(2)S (ref. 4). The reduction of sulphite is a key intermediate step in all sulphate reduction pathways. In Clostridium and Salmonella, an inducible sulphite reductase is directly linked to the regeneration of NAD(+), which has been suggested to have a role in energy production and growth, as well as in the detoxification of sulphite. Above a certain concentration threshold, both H(2)S and HS(-) inhibit cell growth by binding the metal centres of enzymes and cytochrome oxidase, necessitating a release mechanism for the export of this toxic metabolite from the cell. Here we report the identification of a hydrosulphide ion channel in the pathogen Clostridium difficile through a combination of genetic, biochemical and functional approaches. The HS(-) channel is a member of the formate/nitrite transport family, in which about 50 hydrosulphide ion channels form a third subfamily alongside those for formate (FocA) and for nitrite (NirC). The hydrosulphide ion channel is permeable to formate and nitrite as well as to HS(-) ions. Such polyspecificity can be explained by the conserved ion selectivity filter observed in the channel's crystal structure. The channel has a low open probability and is tightly regulated, to avoid decoupling of the membrane proton gradient.     

Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel

5-hydroxytryptamine type 3 (5-HT3) receptors are members of the Cys-loop receptor superfamily. Neurotransmitter binding in these proteins triggers the opening (gating) of an ion channel by means of an as-yet-uncharacterized conformational change. Here we show that a specific proline (Pro 8*), located at the apex of the loop between the second and third transmembrane helices (M2-M3), can link binding to gating through a cis-trans isomerization of the protein backbone. Using unnatural amino acid mutagenesis, a series of proline analogues with varying preference for the cis conformer was incorporated at the 8* position. Proline analogues that strongly favour the trans conformer produced non-functional channels. Among the functional mutants there was a strong correlation between the intrinsic cis-trans energy gap of the proline analogue and the activation of the channel, suggesting that cis-trans isomerization of this single proline provides the switch that interconverts the open and closed states of the channel. Consistent with this proposal, nuclear magnetic resonance studies on an M2-M3 loop peptide reveal two distinct, structured forms. Our results thus confirm the structure of the M2-M3 loop and the critical role of Pro 8* in the 5-HT3 receptor. In addition, they suggest that a molecular rearrangement at Pro 8* is the structural mechanism that opens the receptor pore.     

Location of a delta-subunit region determining ion transport through the acetylcholine receptor channel

The combination of complementary DNA expression and single-channel current analysis provides a powerful tool for studying the structure-function relationship of the nicotinic acetylcholine receptor (AChR) (refs 1-5). We have previously shown that AChR channels consisting of subunits from different species, expressed in the surface membrane of Xenopus oocytes, can be used to relate functional properties to individual subunits. Here we report that, in extracellular solution of low divalent cation concentration, the bovine AChR channel has a smaller conductance than the Torpedo AChR channel. Replacement of the delta-subunit of the Torpedo AChR by the bovine delta-subunit makes the channel conductance similar to that of the bovine AChR channel. To locate the region in the delta-subunit responsible for this difference, we have constructed chimaeric delta-subunit cDNAs with different combinations of the Torpedo and bovine counterparts. The conductances of AChR channels containing chimaeric delta-subunits suggest that a region comprising the putative transmembrane segment M2 and the adjacent bend portion between segments M2 and M3 is involved in determining the rate of ion transport through the open channel.     

Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel

The X-ray structure of a pentameric ligand-gated ion channel from Erwinia chrysanthemi (ELIC) has recently provided structural insight into this family of ion channels at high resolution. The structure shows a homo-pentameric protein with a barrel-stave architecture that defines an ion-conduction pore located on the fivefold axis of symmetry. In this structure, the wide aqueous vestibule that is encircled by the extracellular ligand-binding domains of the five subunits narrows to a discontinuous pore that spans the lipid bilayer. The pore is constricted by bulky hydrophobic residues towards the extracellular side, which probably serve as barriers that prevent the diffusion of ions. This interrupted pore architecture in ELIC thus depicts a non-conducting conformation of a pentameric ligand-gated ion channel, the thermodynamically stable state in the absence of bound ligand. As ligand binding promotes pore opening in these ion channels and the specific ligand for ELIC has not yet been identified, we have turned our attention towards a homologous protein from the cyanobacterium Gloebacter violaceus (GLIC). GLIC was shown to form proton-gated channels that are activated by a pH decrease on the extracellular side and that do not desensitize after activation. Both prokaryotic proteins, ELIC and GLIC form ion channels that are selective for cations over anions with poor discrimination among monovalent cations, characteristics that resemble the conduction properties of the cation-selective branch of the family that includes acetylcholine and serotonin receptors. Here we present the X-ray structure of GLIC at 3.1 A resolution. The structure reveals a conformation of the channel that is distinct from ELIC and that probably resembles the open state. In combination, both structures suggest a novel gating mechanism for pentameric ligand-gated ion channels where channel opening proceeds by a change in the tilt of the pore-forming helices.     

Karyopherin-mediated import of integral inner nuclear membrane proteins

Targeting of newly synthesized integral membrane proteins to the appropriate cellular compartment is specified by discrete sequence elements, many of which have been well characterized. An understanding of the signals required to direct integral membrane proteins to the inner nuclear membrane (INM) remains a notable exception. Here we show that integral INM proteins possess basic sequence motifs that resemble 'classical' nuclear localization signals. These sequences can mediate direct binding to karyopherin-alpha and are essential for the passage of integral membrane proteins to the INM. Furthermore, karyopherin-alpha, karyopherin-beta1 and the Ran GTPase cycle are required for INM targeting, underscoring parallels between mechanisms governing the targeting of integral INM proteins and soluble nuclear transport. We also provide evidence that specific nuclear pore complex proteins contribute to this process, suggesting a role for signal-mediated alterations in the nuclear pore complex to allow for passage of INM proteins along the pore membrane.     

A proton pore in a potassium channel voltage sensor reveals a focused electric field

Voltage-dependent potassium channels are essential for the generation of nerve impulses. Voltage sensitivity is conferred by charged residues located mainly in the fourth transmembrane segment (S4) of each of the four identical subunits that make up the channel. These charged segments relocate when the potential difference across the membrane changes, controlling the ability of the pore to conduct ions. In the crystal structure of the Aeropyrum pernix potassium channel KvAP, the S4 and part of the third (S3B) transmembrane alpha-helices are connected by a hairpin turn in an arrangement termed the 'voltage-sensor paddle'. This structure was proposed to move through the lipid bilayer during channel activation, transporting positive charges across a large fraction of the membrane. Here we show that replacing the first S4 arginine by histidine in the Shaker potassium channel creates a proton pore when the cell is hyperpolarized. Formation of this pore does not support the paddle model, as protons would not have access to a lipid-buried histidine. We conclude that, at hyperpolarized potentials, water and protons from the internal and external solutions must be separated by a narrow barrier in the channel protein that focuses the electric field to a small voltage-sensitive region.     

卫生型泵内部流道流场分析

针对广东日新泵业两台不同参数卫生泵的实测结果,对叶轮内部三维不可压湍流流动进行了数值模拟．通过商用软件Fluent 5．0对两台泵的叶轮进行数值计算,计算过程采用标准k-ε湍流模型及SIMPLEC算法．通过对两台泵流动状况的研究与分析,揭示了叶轮内湍流流动的速度分布、压力分布情况,指出了两台泵叶轮结构的合理性,并提出了第二台泵叶轮的修改方案,同时对泵的水力性能进行了预测,从而为卫生型泵的优化设计奠定了基础．     

X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel

General anaesthetics have enjoyed long and widespread use but their molecular mechanism of action remains poorly understood. There is good evidence that their principal targets are pentameric ligand-gated ion channels (pLGICs) such as inhibitory GABA(A) (γ-aminobutyric acid) receptors and excitatory nicotinic acetylcholine receptors, which are respectively potentiated and inhibited by general anaesthetics. The bacterial homologue from Gloeobacter violaceus (GLIC), whose X-ray structure was recently solved, is also sensitive to clinical concentrations of general anaesthetics. Here we describe the crystal structures of the complexes propofol/GLIC and desflurane/GLIC. These reveal a common general-anaesthetic binding site, which pre-exists in the apo-structure in the upper part of the transmembrane domain of each protomer. Both molecules establish van der Waals interactions with the protein; propofol binds at the entrance of the cavity whereas the smaller, more flexible, desflurane binds deeper inside. Mutations of some amino acids lining the binding site profoundly alter the ionic response of GLIC to protons, and affect its general-anaesthetic pharmacology. Molecular dynamics simulations, performed on the wild type (WT) and two GLIC mutants, highlight differences in mobility of propofol in its binding site and help to explain these effects. These data provide a novel structural framework for the design of general anaesthetics and of allosteric modulators of brain pLGICs.