Altered K+ channel expression in abnormal T lymphocytes from mice with the lpr gene mutation

The observation that voltage-dependent K+ channels are required for activation of human T lymphocytes suggests that pathological conditions involving abnormal mitogen responses might be reflected in ion channel abnormalities. Gigaohm seal techniques were used to study T cells from MRL/MpJ-lpr/lpr mice; these mice develop generalized lymphoproliferation of functionally and phenotypically abnormal T cells and a disease resembling human systemic lupus erythematosus. The number and predominant type of K+ channels in T cells from these mice differ dramatically from those in T cells from control strains and a congenic strain lacking the lpr gene locus. Thus an abnormal pattern of ion channel expression has now been associated with a genetic defect in cells of the immune system.     

A family of three mouse potassium channel genes with intronless coding regions

To understand the molecular mechanisms responsible for generating physiologically diverse potassium channels in mammalian cells, mouse genomic clones have been isolated with a potassium channel complementary DNA, MBK1, that is homologous to the Drosophila potassium channel gene, Shaker. A family of three closely related potassium channel genes (MK1, MK2, and MK3) that are encoded at distinct genomic loci has been isolated. Sequence analysis reveals that the coding region of each of these three genes exists as a single uninterrupted exon in the mouse genome. This organization precludes the generation of multiple forms of the protein by alternative RNA splicing, a mechanism known to characterize the Drosophila potassium channel genes Shaker and Shab. Thus, mammals may use a different strategy for generating diverse K+ channels by encoding related genes at multiple distinct genomic loci, each of which produces only a single protein.     

植物钾(K~+)离子通道的研究

钾在植物生长发育过程中具有许多重要的作用,钾离子通道是植物吸收钾离子的重要途径之一,根据结构和功能的不同钾离子通道可分为Shaker家族通道、KCO通道、其他通道。对上述植物钾离子通道蛋白的生化特性以及结构功能及相关基因研究的进展进行了详细的综述。     

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.     

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.     

A distinct potassium channel polypeptide encoded by the Drosophila eag locus.

Many of the signaling properties of neurons and other electrically excitable cells are determined by a diverse family of potassium channels. A number of genes that encode potassium channel polypeptides have been cloned from various organisms on the basis of their sequence similarity to the Drosophila Shaker (Sh) locus. As an alternative strategy, a molecular analysis of other Drosophila genes that were defined by mutations that perturb potassium channel function was undertaken. Sequence analysis of complementary DNA from the ether à go-go (eag) locus revealed that it encodes a structural component of potassium channels that is related to but is distinct from all identified potassium channel polypeptides.     

Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB

Site-directed mutagenesis experiments have suggested a model for the inactivation mechanism of Shaker potassium channels from Drosophila melanogaster. In this model, the first 20 amino acids form a cytoplasmic domain that interacts with the open channel to cause inactivation. The model was tested by the internal application of a synthetic peptide, with the sequence of the first 20 residues of the ShB alternatively spliced variant, to noninactivating mutant channels expressed in Xenopus oocytes. The peptide restored inactivation in a concentration-dependent manner. Like normal inactivation, peptide-induced inactivation was not noticeably voltage-dependent. Trypsin-treated peptide and peptides with sequences derived from the first 20 residues of noninactivating mutants did not restore inactivation. These results support the proposal that inactivation occurs by a cytoplasmic domain that occludes the ion-conducting pore of the channel.     

Alteration of four identified K+ currents in Drosophila muscle by mutations in eag

Voltage-clamp analysis of Drosophila larval muscle revealed that ether à go-go (eag) mutations affected all identified potassium currents, including those specifically eliminated by mutations in the Shaker or slowpoke gene. Together with DNA sequence analysis, the results suggest that the eag locus encodes a subunit common to different potassium channels. Thus, combinatorial assembly of polypeptides from different genes may contribute to potassium channel diversity.     

Molecular architecture of the KvAP voltage-dependent K+ channel in a lipid bilayer

We have analyzed the local structure and dynamics of the prokaryotic voltage-dependent K+ channel (KvAP) at 0 millivolts, using site-directed spin labeling and electron paramagnetic resonance spectroscopy. We show that the S4 segment is located at the protein/lipid interface, with most of its charges protected from the lipid environment. Structurally, S4 is highly dynamic and is separated into two short helices by a flexible linker. Accessibility and dynamics data indicate that the S1 segment is surrounded by other parts of the protein. We propose that S1 is at the contact interface between the voltage-sensing and pore domains. These results establish the general principles of voltage-dependent channel structure in a biological membrane.     

Sequence and expression of mRNAs encoding the alpha 1 and alpha 2 subunits of a DHP-sensitive calcium channel

Complementary DNAs were isolated and used to deduce the primary structures of the alpha 1 and alpha 2 subunits of the dihydropyridine-sensitive, voltage-dependent calcium channel from rabbit skeletal muscle. The alpha 1 subunit, which contains putative binding sites for calcium antagonists, is a hydrophobic protein with a sequence that is consistent with multiple transmembrane domains and shows structural and sequence homology with other voltage-dependent ion channels. In contrast, the alpha 2 subunit is a hydrophilic protein without homology to other known protein sequences. Nucleic acid hybridization studies suggest that the alpha 1 and alpha 2 subunit mRNAs are expressed differentially in a tissue-specific manner and that there is a family of genes encoding additional calcium channel subtypes.     

Expression of a cloned rat brain potassium channel in Xenopus oocytes

Potassium channels are ubiquitous membrane proteins with essential roles in nervous tissue, but little is known about the relation between their function and their molecular structure. A complementary DNA library was made from rat hippocampus, and a complementary DNA clone (RBK-1) was isolated. The predicted sequence of the 495-amino acid protein is homologous to potassium channel proteins encoded by the Shaker locus of Drosophila and differs by only three amino acids from the expected product of a mouse clone MBK-1. Messenger RNA transcribed from RBK-1 in vitro directed the expression of potassium channels when it was injected into Xenopus oocytes. The potassium current through the expressed channels resembles both the transient (or A) and the delayed rectifier currents reported in mammalian neurons and is sensitive to both 4-aminopyridine and tetraethylammonium.     

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.     

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.     

K+ current diversity is produced by an extended gene family conserved in Drosophila and mouse

The Drosophila Shaker gene on the X chromosome has three sister genes, Shal, Shab, and Shaw, which map to the second and third chromosomes. This extended gene family encodes voltage-gated potassium channels with widely varying kinetics (rate of macroscopic current activation and inactivation) and voltage sensitivity of steady-state inactivation. The differences in the currents of the various gene products are greater than the differences produced by alternative splicing of the Shaker gene. In Drosophila, the transient (A current) subtype of the potassium channel (Shaker and Shal) and the delayed-rectifier subtype (Shab and Shaw) are encoded by homologous genes, and there is more than one gene for each subtype of channel. Homologs of Shaker, Shal, Shab, and Shaw are present in mammals; each Drosophila potassium-channel gene may be represented as a multigene subfamily in mammals.     

Atrotoxin: a specific agonist for calcium currents in heart

A specific label for voltage-dependent calcium channels is essential for the isolation and purification of the membrane protein that constitutes the calcium channel and for a better understanding of its function. A fraction of Crotalus atrox that increases voltage-dependent calcium currents in single, dispersed guinea pig ventricular cells was isolated. In the doses used, neither sodium nor potassium currents were changed. The fraction was active in the absence of detectable phospholipase or protease activity, and the active component, designated atrotoxin, produced its effect rapidly and reversibly. The effect was produced by extracellular but not intracellular application of the agent. The increase in Ca2+ current was blocked by the Ca2+ channel blockers cobalt and nitrendipine. The active fraction completely blocked specific [3H]nitrendipine binding to guinea pig ventricular membrane preparations. The inhibition of nitrendipine binding by atrotoxin was apparently via an allosteric mechanism. Thus atrotoxin was shown to bind to the Ca2+ channel and to act as a specific Ca2+ channel agonist.     

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.     

Single-channel and genetic analyses reveal two distinct A-type potassium channels in Drosophila

Whole-cell and single-channel voltage-clamp techniques were used to identify and characterize the channels underlying the fast transient potassium current (A current) in cultured myotubes and neurons of Drosophila. The myotube (A1) and neuronal (A2) channels are distinct, differing in conductance, voltage dependence, and gating kinetics. The myotube currents have a faster and more voltage-dependent macroscopic inactivation rate, a larger steady-state component, and a less negative steady-state inactivation curve than the neuronal currents. The myotube channels have a conductance of 12 to 16 picosiemens, whereas the neuronal channels have a conductance of 5 to 8 picosiemens. In addition, the myotube channel is affected by Shaker mutations, whereas the neuronal channel is not. Together, these data suggest that the two channels are separate molecular structures, the expression of which is controlled, at least in part, by different genes.     

Ion selectivity in a semisynthetic K+ channel locked in the conductive conformation

Potassium channels are K+-selective protein pores in cell membrane. The selectivity filter is the functional unit that allows K+ channels to distinguish potassium (K+) and sodium (Na+) ions. The filter's structure depends on whether K+ or Na+ ions are bound inside it. We synthesized a K+ channel containing the d-enantiomer of alanine in place of a conserved glycine and found by x-ray crystallography that its filter maintains the K+ (conductive) structure in the presence of Na+ and very low concentrations of K+. This channel conducts Na+ in the absence of K+ but not in the presence of K+. These findings demonstrate that the ability of the channel to adapt its structure differently to K+ and Na+ is a fundamental aspect of ion selectivity, as is the ability of multiple K+ ions to compete effectively with Na+ for the conductive filter.