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ScienceWeek
CELL PHYSIOLOGY: ON VOLTAGE-GATED ION CHANNELS
The following points are made by Z. Sands et al (Current Biology 2005 15:R44):
1) Voltage-gated ion channels are integral membrane proteins that enable the passage of selected inorganic ions across cell membranes. They open and close in response to changes in transmembrane voltage, and play a key role in electrical signaling by excitable cells such as neurons. Voltage-gated K+, Na+ and Ca2+ channels are thought to have similar overall architectures. X-ray crystallographic studies of bacterial homologs have provided considerable insights into the relationship between channel structure and function in various classes of K+ channels, including the voltage-gated (Kv) ones. But despite these advances, the exact structure of the Kv voltage sensor, and how the Kv channel structure changes in response to changes in transmembrane voltage, remain elusive.
2) When the cell membrane is polarized, so that the interior of the cell is at a negative voltage relative to the exterior, Kv channels remain closed. When the membrane is depolarized, these channels open rapidly (less than 1 ms), allowing ions to flow passively down their electrochemical gradients, at near diffusion rates. Kv channels thus have two principal functions: ion conduction, and voltage sensing. Corresponding to these two functions, Kv channel subunits contain two distinct, but functionally coupled transmembrane domains. The pore domain is responsible for the ion selectivity and conduction, and also for channel gating per se, whereas the voltage-sensing domain triggers a change in conformation of the pore domain in response to changes in transmembrane voltage.
3) Kv channels comprise four subunits that encircle a central ion conduction pathway. Each subunit consists of six a helices (S1-S6) with both amino and carboxyl termini on the intracellular side of the membrane. The first four transmembrane helices (S1-S4) form the voltage-sensing domain, whereas the last two transmembrane helices (S5-S6), along with an intervening re-entrant P loop, form the pore domain. The re-entrant loop contains a short pore helix and an extended region of polypeptide chain that contains the characteristic sequence motif TVGYG and forms the selectivity filter.
4) The filter region forms the extracellular end of the pore. The TVGYG motif is highly conserved amongst K+ channels. The glycine residues of this motif enable the filter to adopt a conformation in which the mainchain carbonyl oxygen atoms point toward the center of the pore axis, generating five discrete binding sites for K+ ions flowing through the pore. The filter region exhibits a degree of flexibility which may be responsible for "fast gating" of K+ channels [1]. The main activation gate, however, lies at the opposite end of the channel, at its cytoplasmic mouth.
5) The ion conduction pathway can switch between two main functional states, open and closed. The structural differences between these two states have been revealed by comparing the X-ray structures of KcsA, crystallised in a closed conformation, and of MthK, crystallised in an open conformation. This comparison [2] suggested that bending or kinking of the inner pore-lining helices -- M2 in KcsA and MthK, S6 in Kv channels --plays a key role in pore gating. In KcsA, the M2 helices are undistorted, and converge to form a narrow hydrophobic constriction near the cytoplasmic entry to the pore. In contrast, in MthK or the bacterial voltage gated channel KvAP, the inner helices bend at a conserved glycine residue and so splay apart so as to open up the intracellular mouth.[3-5]
References (abridged):
1. Domene, C., Grottesi, A. and Sansom, M.S.P. (2004). Filter flexibility and distortion in a bacterial inward rectifier K+ channel: simulation studies of KirBac1.1. Biophys. J. 87, 256-267
2. Jiang, Y., Lee, A., Chen, J., Cadene, M., Chait, B.T. and MacKinnon, R. (2002). The open pore conformation of potassium channels. Nature 417, 523-526
3. Labro, A.J., Raes, A.L., Bellens, I., Ottschytsch, N. and Snyders, D.J. (2003). Gating of Shaker-type channels requires the flexibility of S6 caused by prolines. J. Biol. Chem. 278, 50724-50731
4. Jiang, Y., Lee, A., Chen, J., Ruta, V., Cadene, M., Chait, B.T. and Mackinnon, R. (2003). X-ray structure of a voltage-dependent K+ channel. Nature 423, 33-41
5. Gandhi, C.C. and Isacoff, E.Y. (2002). Molecular models of voltage sensing. J. Gen. Physiol. 120, 455-463
Current Biology http://www.current-biology.com
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Related Material:
STRUCTURE OF THE VOLTAGE-GATED SODIUM CHANNEL.
Notes by ScienceWeek:
Electrical signals in the nervous system are generated by the movement of ions across the nerve cell membrane. These ionic currents flow through aqueous pores of membrane proteins known as "ion channels", and these channels vary in ion selectivity, with some channels specifically permeable to sodium or potassium or calcium ions.
Certain sodium, potassium, and calcium channels are "voltage-gated" -- their permeability is switched on and off by changes in the potential difference across the cell membrane --and these channels essentially control the dynamic electric activity of nerve and muscle cells.
The voltage-activated sodium channel from eel electric organs (of relevance in this report) is a single molecule of approximately 1800 amino acids, within which are 4 repeating domains. These domains are architecturally equivalent to the subunits of other ion channels. Within each domain, there are apparently 6 membrane spanning regions connected by intracellular and extracellular polypeptide loops. The eel channel is apparently representative of a diverse family of channel proteins present in nerve and muscle fibers. Fifty years ago, the existence of such specific protein channels was not even conceived, although it was recognized there had to be some explanation for specific ion currents in nerve and muscle cells. Thirty years ago, the existence of such channels was vaguely proposed, but with hardly any molecular information. In the past two decades, ion channels have become definitive structural entities.
The following points are made by C. Sato et al (Nature 2001 409:1047):
1) The authors point out that the voltage-sensitive sodium, potassium, and calcium channels operate together to amplify, transmit, and generate electric pulses in animals. Sodium and calcium channels are involved in cell excitation, neuronal transmission, muscle contraction, and many functions that relate directly to human diseases. Sodium channels, which are glycosylated proteins with a relative molecular mass of approximately 300,000, are responsible for signal transduction and amplification, and are chief targets of anesthetic drugs and neurotoxins.
2) The authors report the 3-dimensional structure of the voltage-sensitive sodium channel of the eel Electrophorus electricus. The structure was determined at 19 angstroms resolution by helium-cooled cryo-electron microscopy and single-particle image analysis of the solubilized sodium channel. The authors report the channel has a bell-shaped outer surface of 135 angstroms in height and 100 angstroms in side length at the square-shaped bottom, and a spherical top with a diameter of 65 angstroms. Several inner cavities are connected to 4 small holes and 8 orifices close to the extracellular and cytoplasmic membrane surfaces. Homologous voltage-sensitive calcium and tetrameric potassium channels, which regulate secretory processes and the membrane potential, may possess related structure.
Nature http://www.nature.com/nature
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Related Material:
NEUROBIOLOGY: ATOMIC SCALE MOVEMENTS IN POTASSIUM CHANNELS.
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Notes by ScienceWeek:
The functional electrical activity of nerve cells is based essentially on the rapid movements of ions across the membranes of these cells, especially the movements of sodium and potassium ions. These ion movements occur through special pores ("ion channels") in the cell membrane, and one of the important problems during the past few decades has been to characterize these ion channels at the molecular level. Most ion channels are selective, allowing only ions of a certain type to pass, and an individual cell has ion channels with various ion selectivities. The selectivity of an ion channel can be "gated", the channel effectively opened or closed, and ion channels are said to "voltage-gated" or "ligand-gated", depending on how the change in selectivity is provoked. The term "voltage-gated" refers to the opening or closing of an ion channel by changes in the electrical potential across the membrane, while the term "ligand-gated" refers to opening and closing of an ion channel by interactions between ligands and membrane receptors.
It has become apparent that voltage-gated ion channels are transmembrane proteins consisting of 4 identical subunits, each of which contains 6 transmembrane segments. Studies of the potassium ion channel have identified two segments that contain several charged protein residues, and these charged residues apparently sense changes in the potential difference across the membrane and form part of the membrane "voltage sensor". Although these regions apparently undergo conformational changes in response to changes in membrane potential, little is known about the nature of these changes.
The following points are made by A. Cha et al ((Nature 1999 402:809):
1) The authors used *lanthanide-based fluorescence resonance energy transfer to measure distances between *Shaker potassium-channel protein subunits at specific residues. Voltage dependent distance changes of up to 3.2 angstroms were measured at several sites near one of the charged protein segments (S4). These movements directly correlated with electrical measurements of the voltage sensor, establishing a link between physical changes and electrical charge movement.
2) The authors suggest that the measured distance changes indicate that the region associated with the S4 segment undergoes a rotation and possible tilt, rather than a large transmembrane movement, in response to voltage.
3) The authors conclude: "These results demonstrate the first in situ measurement of atomic scale movement in a transmembrane protein."
In a contiguous and related report, K.S. Glauner et al (Nature 1999 402:813) make the following points:
1) The authors used fluorescence resonance energy transfer as a "spectroscopic ruler" to determine distances between S4 subunits in the Shaker potassium channel in different gating states.
2) The authors conclude their experimental evidence is consistent with the S4 subunit being a tilted helix that twists during activation. The authors propose that helical twist contributes to the movement of charged side chains across the membrane electric field and that this movement is involved in coupling voltage-sensing to gating.
Nature http://www.nature.com/nature
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Notes by ScienceWeek:
lanthanide-based fluorescence resonance energy transfer: The term "fluorescence resonance energy transfer" (also called "fluorescence energy transfer") refers to energy transfer between two fluorophores (chemical groups or molecules capable of fluorescence). If the two fluorophores are attached to a molecule at different positions, observations of fluorescence energy transfer between them can be used to determine the distance between the two attachment positions. Lanthanide-based resonance energy transfer is a modification of conventional fluorescence resonance energy transfer in which a long-lived lanthanide donor transfers energy in a distance-dependent manner to a conventional organic fluorescent acceptor. This technique has previously been used to measure angstrom-scale conformational changes in proteins.
Shaker potassium-channel protein: The term "Shaker" here refers to a mutant of the fruit fly Drosophila, the mutant exhibiting intense shaking of the legs and body in response to exposure to a volatile anesthetic. Genetic analysis of the mutation some years ago led to the sequencing of a Drosophila gene expressing a potassium-channel protein, the shaking of the insect apparently resulting from a mutation in this gene, with the mutation producing long-lasting potassium ion currents when nerve fibers are activated. Once the Shaker gene was sequenced, a conventional procedure was developed in the 1980s to have this gene expressed in frog egg cells (oocytes) in order to advance the study of the behavior of potassium ion channels. Thus, the potassium ion channels in this report are ion channels derived from the fruit fly Drosophila and expressed in the frog Xenopus laevis egg cell. The essential aspects of frog oocytes is that they are large cells (up to 1 millimeter in diameter), and the introduction of foreign *messenger RNA into the egg cell readily results in the production of the protein encoded by the introduced messenger RNA.
messenger RNA: (mRNA) The ribonucleic acid molecule transcribed from DNA that carries the coded information specifying the sequence of amino acids in a protein.
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