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.     

Biophysical and molecular mechanisms of Shaker potassium channel inactivation

The potassium channels encoded by the Drosophila Shaker gene activate and inactivate rapidly when the membrane potential becomes more positive. Site-directed mutagenesis and single-channel patch-clamp recording were used to explore the molecular transitions that underlie inactivation in Shaker potassium channels expressed in Xenopus oocytes. A region near the amino terminus with an important role in inactivation has now been identified. The results suggest a model where this region forms a cytoplasmic domain that interacts with the open channel to cause inactivation.     

Molecular basis of gating charge immobilization in Shaker potassium channels

Voltage-dependent ion channels respond to changes in the membrane potential by means of charged voltage sensors intrinsic to the channel protein. Changes in transmembrane potential cause movement of these charged residues, which results in conformational changes in the channel. Movements of the charged sensors can be detected as currents known as gating currents. Measurement of the gating currents of the Drosophila Shaker potassium channel indicates that the charge on the voltage sensor of the channels is progressively immobilized by prolonged depolarizations. The charge is not immobilized in a mutant of the channel that lacks inactivation. These results show that the region of the molecule responsible for inactivation interacts, directly or indirectly, with the voltage sensor to prevent the return of the charge to its original position. The gating transitions between closed states of the channel appear not to be independent, suggesting that the channel subunits interact during activation.     

Regulation of chloride channels by protein kinase C in normal and cystic fibrosis airway epithelia

Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.     

Structure of the cytoplasmic beta subunit-T1 assembly of voltage-dependent K+ channels

The structure of the cytoplasmic assembly of voltage-dependent K+ channels was solved by x-ray crystallography at 2.1 angstrom resolution. The assembly includes the cytoplasmic (T1) domain of the integral membrane alpha subunit together with the oxidoreductase beta subunit in a fourfold symmetric T1(4)beta4 complex. An electrophysiological assay showed that this complex is oriented with four T1 domains facing the transmembrane pore and four beta subunits facing the cytoplasm. The transmembrane pore communicates with the cytoplasm through lateral, negatively charged openings above the T1(4)beta4 complex. The inactivation peptides of voltage-dependent K(+) channels reach their site of action by entering these openings.     

Activity- and mTOR-dependent suppression of Kv1.1 channel mRNA translation in dendrites

Mammalian target of rapamycin (mTOR) is implicated in synaptic plasticity and local translation in dendrites. We found that the mTOR inhibitor, rapamycin, increased the Kv1.1 voltage-gated potassium channel protein in hippocampal neurons and promoted Kv1.1 surface expression on dendrites without altering its axonal expression. Moreover, endogenous Kv1.1 mRNA was detected in dendrites. Using Kv1.1 fused to the photoconvertible fluorescence protein Kaede as a reporter for local synthesis, we observed Kv1.1 synthesis in dendrites upon inhibition of mTOR or the N-methyl-d-aspartate (NMDA) glutamate receptor. Thus, synaptic excitation may cause local suppression of dendritic Kv1 channels by reducing their local synthesis.     

Identification of an intracellular peptide segment involved in sodium channel inactivation

Antibodies directed against a conserved intracellular segment of the sodium channel alpha subunit slow the inactivation of sodium channels in rat muscle cells. Of four site-directed antibodies tested, only antibodies against the short intracellular segment between homologous transmembrane domains III and IV slowed inactivation, and their effects were blocked by the corresponding peptide antigen. No effects on the voltage dependence of sodium channel activation or of steady-state inactivation were observed, but the rate of onset of the antibody effect and the extent of slowing of inactivation were voltage-dependent. Antibody binding was more rapid at negative potentials, at which sodium channels are not inactivated; antibody-induced slowing of inactivation was greater during depolarizations to more positive membrane potentials. The peptide segment recognized by this antibody appears to participate directly in rapid sodium channel inactivation during large depolarizations and to undergo a conformational change that reduces its accessibility to antibodies as the channel inactivates.     

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.     

MA包装与1-MCP处理对苹果的贮藏效应研究

以藤牧一号苹果为试材,研究(0±1)℃贮藏条件下气调保鲜包装(MA包装)与1-甲基环丙烯(1-MCP)处理对果实采后生理的影响。结果表明:1-MCP结合MA包装处理可显著抑制贮藏期间果实呼吸跃变峰的出现及呼吸速率的上升,减缓果肉硬度下降,延缓丙二醛含量和相对膜透性的升高,从而降低膜脂过氧化程度。短期(0±1)℃贮存条件下,藤牧一号苹果适宜选择0.02 mm或者0.03 mm MA包装,长期(0±1)℃贮存则适宜使用0.05 mm MA包装。     

Acetylcholine and GABA mediate opposing actions on neuronal chloride channels in crayfish

A central principle of neural integration is that excitatory and inhibitory neurotransmitters effect the opening of distinct classes of membrane ionic channels and that integration consists of the summation of the opposing ionic currents on the postsynaptic membrane. In tangential cells of crayfish optic lobes, a hyperpolarizing, biphasic synaptic potential is produced by the concurrent action of acetylcholine and gamma aminobutyric acid (GABA). Acetylcholine hyperpolarizes the cell and increases chlorine conductance. GABA depolarizes the cell by closing some of the same chloride channels. Therefore, in this case integration is achieved by the antagonistic actions of two transmitters on the same ionic channel.     

Ca2+ and Ca2+-activated K+ currents in mammalian gastric smooth muscle cells

Inward movement of calcium through voltage-dependent channels in muscle is thought to initiate the action potential and trigger contraction. Calcium-activated potassium channels carry large outward potassium currents that may be responsible for membrane repolarization. Calcium and calcium-activated potassium currents were identified in enzymatically isolated mammalian gastric myocytes. These currents were blocked by cadmium and nifedipine but were not substantially affected by diltiazem or D600. No evidence for a tetrodotoxin-sensitive sodium current or an inwardly rectifying potassium current was found.     

Graded regulation of the Kv2.1 potassium channel by variable phosphorylation

Dynamic modulation of ion channels by phosphorylation underlies neuronal plasticity. The Kv2.1 potassium channel is highly phosphorylated in resting mammalian neurons. Activity-dependent Kv2.1 dephosphorylation by calcineurin induces graded hyperpolarizing shifts in voltage-dependent activation, causing suppression of neuronal excitability. Mass spectrometry-SILAC (stable isotope labeling with amino acids in cell culture) identified 16 Kv2.1 phosphorylation sites, of which 7 were dephosphorylated by calcineurin. Mutation of individual calcineurin-regulated sites to alanine produced incremental shifts mimicking dephosphorylation, whereas mutation to aspartate yielded equivalent resistance to calcineurin. Mutations at multiple sites were additive, showing that variable phosphorylation of Kv2.1 at a large number of sites allows graded activity-dependent regulation of channel gating and neuronal firing properties.     

Gating currents of the sodium channels: three ways to block them

Preceding the opening of the sodium channels of axon membrane there is a small outward current, gating current, that is probably associated with the molecular rearrangements that open the channels. Gating current is reversibly blocked by three procedures that block the sodium current: (i) internal perfusion with zinc ions, (ii) inactivation of sodium conductance by brief depolarization, and (iii) prolonged depolarization.     

HCN2 ion channels play a central role in inflammatory and neuropathic pain

The rate of action potential firing in nociceptors is a major determinant of the intensity of pain. Possible modulators of action potential firing include the HCN ion channels, which generate an inward current, I(h), after hyperpolarization of the membrane. We found that genetic deletion of HCN2 removed the cyclic adenosine monophosphate (cAMP)-sensitive component of I(h) and abolished action potential firing caused by an elevation of cAMP in nociceptors. Mice in which HCN2 was specifically deleted in nociceptors expressing Na(V)1.8 had normal pain thresholds, but inflammation did not cause hyperalgesia to heat stimuli. After a nerve lesion, these mice showed no neuropathic pain in response to thermal or mechanical stimuli. Neuropathic pain is therefore initiated by HCN2-driven action potential firing in Na(V)1.8-expressing nociceptors.     

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.     

1-MCP处理对"美国8号"苹果采后生理和相关酶活性的影响

以"美国8号"苹果为试材,研究了0℃贮藏条件下1-MCP处理对采后生理及相关酶活性的影响,结果表明:1μl/L 1-MCP处理可以显著抑制贮藏期间果实呼吸速率和乙烯释放量,延缓两者跃变高峰的出现;减缓果肉硬度和可滴定酸含量的下降,但对可溶性固形物含量无明显影响;同时保持贮藏后期SOD、POD、CAT的较高活性,从而降低膜脂过氧化程度,延缓果实衰老.     

Development of excitability in embryonic muscle cell membranes in certain tunicates

During the course of development of muscle cells in certain tunicates, a sign of regenerative membrane response appears in the gastrula stage. In the early tadpole larva, the action potential consists of a spike followed by a plateau. The latter-disappears in fully differentiated cells, conceivably in association with the establishment of delayed rectification.     

6-BA对切花菊瓶插期间膜透性等生理效应的调节

探讨了瓶插期间6-BA延缓切花菊衰老的机理。结果发现,6-BA预处理抑制了膜透性的增大;过氧化产物丙二醛(MDA)的产生得到抑制;并且与衰老相关的过氧化物酶(POD)和超氧化物歧化酶(SOD)的活性分别得到抑制和提高。     

Voltage-dependent sodium channels in an invertebrate striated muscle

Striated skeletal muscles from the planktonic arrowworm Sagitta elegans (phylum Chaetognatha) were voltage-clamped. The muscles displayed classical voltage-dependent sodium channels that (i) showed peak transient currents when the membrane was depolarized 90 millivolts from rest, (ii) opened rapidly with peak currents flowing within 0.4 milliseconds at 4 degrees C, (iii) showed voltage-dependent inactivation with 50 percent inactivation at +25 millivolts from rest, and (iv) were blocked by 500 nanomolar tetrodotoxin.