The potassium channel from Streptomyces lividans is an integral membrane protein with sequence similarity to all known K+ channels, particularly in the pore region. X-ray analysis with data to 3.2 angstroms reveals that four identical subunits create an inverted teepee, or cone, cradling the selectivity filter of the pore in its outer end. The narrow selectivity filter is only 12 angstroms long, whereas the remainder of the pore is wider and lined with hydrophobic amino acids. A large water-filled cavity and helix dipoles are positioned so as to overcome electrostatic destabilization of an ion in the pore at the center of the bilayer. Main chain carbonyl oxygen atoms from the K+ channel signature sequence line the selectivity filter, which is held open by structural constraints to coordinate K+ ions but not smaller Na+ ions. The selectivity filter contains two K+ ions about 7.5 angstroms apart. This configuration promotes ion conduction by exploiting electrostatic repulsive forces to overcome attractive forces between K+ ions and the selectivity filter. The architecture of the pore establishes the physical principles underlying selective K+ conduction.

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The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and Selectivity

During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism’s pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.

Bacillus thuringiensis and Its Pesticidal Crystal Proteins

Regulated Translation Initiation Controls Stress-Induced Gene Expression in Mammalian Cells

Acidic amino acids are putative excitatory synaptic transmitters1,2, the ionic mechanism of which is not well understood. Recent studies with selective agonists and antagonists suggest that neurones of the mammalian central nervous system possess several different receptors for acidic amino acids3,4, which in turn are coupled to separate conductance mechanisms5. N-methyl-D-aspartic acid (NMDA) is a selective agonist for one of these receptors3,4. The excitatory action of amino acids acting at NMDA receptors is remarkably sensitive to the membrane potential and it has been suggested that the NMDA receptor is coupled to a voltage-sensitive conductance6–9. Recently, patch-clamp experiments have shown the voltage-dependent block by Mg2+ of current flow through ion channels activated by L-glutamate10. We now show using voltage-clamp experiments on mouse spinal cord neurones that the voltage-sensitivity of NMDA action is greatly reduced on the withdrawal of physiological concentrations (∼1 mM) of Mg2+ from the extracellular fluid. This provides further evidence that Mg2+ blocks inward current flow through ion channels linked to NMDA receptors.

Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones.

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.

http://science.sciencemag.org/content/sci/309/5736/897.full.pdf

Crystal Structure of a Mammalian Voltage-Dependent Shaker Family K + Channel

Charybdotoxin, a protein inhibitor of single Ca2+-activated K+ channels from mammalian skeletal muscle

ClC Cl- channels make up a large molecular family, ubiquitous with respect to both organisms and cell types. In eukaryotes, these channels fulfill numerous biological roles requiring gated anion conductance, from regulating skeletal muscle excitability to facilitating endosomal acidification by (H+)ATPases. In prokaryotes, ClC functions are unknown except in Escherichia coli, where the ClC-ec1 protein promotes H+ extrusion activated in the extreme acid-resistance response common to enteric bacteria. Recently, the high-resolution structure of ClC-ec1 was solved by X-ray crystallography. This primal prokaryotic ClC structure has productively guided understanding of gating and anion permeation in the extensively studied eukaryotic ClC channels. We now show that this bacterial homologue is not an ion channel, but rather a H+-Cl- exchange transporter. As the same molecular architecture can support two fundamentally different transport mechanisms, it seems that the structural boundary separating channels and transporters is not as clear cut as generally thought.

Secondary active transport mediated by a prokaryotic homologue of ClC Cl - channels

I Basics- 1 The Physical Nature of Planar Bilayer Membranes- 2 Ion Channel Electrostatics and the Shapes of Channel Proteins- 3 Superoxide Dismutase as a Model Ion Channel- 4 Single-Channel Enzymology- 5 How to Set Up a Bilayer System- 6 Fusion of Liposomes to Planar Bilayers- 7 Incorporation of Ion Channels by Fusion- II Nicotinic Acetylcholine Receptor- 8 The Reconstituted Acetylcholine Receptor- 9 Immunologic Analysis of the Acetylcholine Receptor- 10 Function of Acetylcholine Receptors in Reconstituted Liposomes- III Sodium Channel- 11 Skeletal Muscle Sodium Channels: Isolation and Reconstitution- 12 Reconstitution of the Sodium Channel from Electrophorus electricus- 13 The Reconstituted Sodium Channel from Brain- 14 Gating of Batrachotoxin-Activated Sodium Channels in Lipid Bilayers- 15 Ion Conduction Through Sodium Channels in Planar Lipid Bilayers- 16 Blocking Pharmacology of Batrachotoxin-Activated Sodium Channels- IV Other Channels in Model Membranes- 17 The Large Calcium-Activated Potassium Channel- 18 The Sarcoplasmic Reticulum Potassium Channel: Lipid Effects- 19 Characterization of Dihydropyridine-Sensitive Calcium Channels from Purified Skeletal Muscle Transverse Tubules- 20 Calcium Channels- 21 Phosphorylation of a Reconstituted Potassium Channel- 22 Voltage Gating in VDAC: Toward a Molecular Mechanism- 23 Analysis and Chemical Modification of Bacterial Porins

Ion Channel Reconstitution

Chloride channels from Torpedo californica electroplax were inserted into planar phospholipid membranes, and single-channel currents were studied at high time-resolution. The open channel fluctuates rapidly between three substates, with conductances of 18.5, 9.4 and 0 pS in 150 mM Cl-. Under various ionic conditions the three substates are always equally spaced in conductance; at various voltages leading to different probabilities of observing the three substates, the substate frequencies are always binomially distributed. The conclusion emerges that the conducting of unit of Cl- channel is composed of two identical Cl- diffusion pathways, each with a voltage-dependent gate.

Open-State Substructure of Single Chloride Channels from Torpedo Electroplax

The mechanism of charybdotoxin (CTX) block of single Ca2+-activated K+ channels from rat muscle was studied in planar lipid bilayers. CTX blocks the channel from the external solution, and K+ in the internal solution specifically relieves toxin block. The effect of K+ is due solely to an enhancement of the CTX dissociation rate. As internal K+ is raised, the CTX dissociation rate increases in a rectangular hyperbolic fashion from a minimum value at low K+ of 0.01 s-1 to a maximum value of approximately 0.2 s-1. As the membrane is depolarized, internal K+ more effectively accelerates CTX dissociation. As the membrane is hyperpolarized, the toxin dissociation rate approaches 0.01 s-1, regardless of the K+ concentration. When internal K+ is replaced by Na+, CTX dissociation is no longer voltage dependent. The permeant ion Rb also accelerates toxin dissociation from the internal solution, while the impermeant ions Li, Na, Cs, and arginine do not. These results argue that K ions can enter the CTX-blocked channel from the internal solution to reach a site located nearly all the way through the conduction pathway; when K+ occupies this site, CTX is destabilized on its blocking site by approximately 1.8 kcal/mol. The most natural way to accommodate these conclusions is to assume that CTX physically plugs the channel's externally facing mouth.

https://rupress.org/jgp/article-pdf/91/3/335/1247511/335.pdf

Christopher Miller

Papers

Charybdotoxin, a protein inhibitor of single Ca2+-activated K+ channels from mammalian skeletal muscle

Secondary active transport mediated by a prokaryotic homologue of ClC Cl - channels

Ion Channel Reconstitution

Open-State Substructure of Single Chloride Channels from Torpedo Electroplax

Mechanism of charybdotoxin block of the high-conductance, Ca2+-activated K+ channel.