Mechanism of magnesium activation of calcium-activated potassium channels

Large-conductance (BK type) Ca(2+)-dependent K(+) channels are essential for modulating muscle contraction and neuronal activities such as synaptic transmission and hearing. BK channels are activated by membrane depolarization and intracellular Ca(2+) and Mg(2+) (refs 6-10). The energy provided by voltage, Ca(2+) and Mg(2+) binding are additive in activating the channel, suggesting that these signals open the activation gate through independent pathways. Here we report a molecular investigation of a Mg(2+)-dependent activation mechanism. Using a combined site-directed mutagenesis and structural analysis, we demonstrate that a structurally new Mg(2+)-binding site in the RCK/Rossman fold domain -- an intracellular structural motif that immediately follows the activation gate S6 helix -- is responsible for Mg(2+)-dependent activation. Mutations that impair or abolish Mg(2+) sensitivity do not affect Ca(2+) sensitivity, and vice versa. These results indicate distinct structural pathways for Mg(2+)- and Ca(2+)-dependent activation and suggest a possible mechanism for the coupling between Mg(2+) binding and channel opening.     

Histamine and noradrenaline decrease calcium-activated potassium conductance in hippocampal pyramidal cells

Ample evidence exists for histaminergic and noradrenergic projections to the hippocampus. Both amines exert neurotransmitter or modulator actions on principal neurones in the CA 1 and in the dentate area. A number of mechanisms have been proposed for these actions, including increased potassium conductance, increased chloride conductance and electrogenic pump stimulation, and reduction of the anomalous inward rectification. Action potentials, and particularly bursts of spikes, in CA 1 pyramidal cells, are followed by an afterhyperpolarization (AHP) which consists of two components. The late AHP depends on a calcium-activated potassium conductance gK+ (Ca2+), and has recently been shown to be increased by dopamine. We report here a rapid and reversible decrease of the late AHP component following a burst of sodium spikes or a calcium spike, during perfusion with micromolar concentrations of histamine and noradrenaline. This effect is mediated by H2 receptors and beta-receptors, respectively, and occurred in the absence of changes in the calcium spike. By such a mechanism histamine and noradrenaline can profoundly potentiate the excitatory impact of depolarizing signals.     

Vasoregulation by the beta1 subunit of the calcium-activated potassium channel

Small arteries exhibit tone, a partially contracted state that is an important determinant of blood pressure. In arterial smooth muscle cells, intracellular calcium paradoxically controls both contraction and relaxation. The mechanisms by which calcium can differentially regulate diverse physiological responses within a single cell remain unresolved. Calcium-dependent relaxation is mediated by local calcium release from the sarcoplasmic reticulum. These 'calcium sparks' activate calcium-dependent potassium (BK) channels comprised of alpha and beta1 subunits. Here we show that targeted deletion of the gene for the beta1 subunit leads to a decrease in the calcium sensitivity of BK channels, a reduction in functional coupling of calcium sparks to BK channel activation, and increases in arterial tone and blood pressure. The beta1 subunit of the BK channel, by tuning the channel's calcium sensitivity, is a key molecular component in translating calcium signals to the central physiological function of vasoregulation.     

Enzymatic activation of voltage-gated potassium channels

Voltage-gated ion channels in excitable nerve, muscle, and endocrine cells generate electric signals in the form of action potentials. However, they are also present in non-excitable eukaryotic cells and prokaryotes, which raises the question of whether voltage-gated channels might be activated by means other than changing the voltage difference between the solutions separated by the plasma membrane. The search for so-called voltage-gated channel activators is motivated in part by the growing importance of such agents in clinical pharmacology. Here we report the apparent activation of voltage-gated K+ (Kv) channels by a sphingomyelinase.     

hERG potassium channels and cardiac arrhythmia

hERG potassium channels are essential for normal electrical activity in the heart. Inherited mutations in the HERG gene cause long QT syndrome, a disorder that predisposes individuals to life-threatening arrhythmias. Arrhythmia can also be induced by a blockage of hERG channels by a surprisingly diverse group of drugs. This side effect is a common reason for drug failure in preclinical safety trials. Insights gained from the crystal structures of other potassium channels have helped our understanding of the block of hERG channels and the mechanisms of gating.     

Localized Ca2+ and calcium-activated potassium conductances in terminals of a barnacle photoreceptor

Calcium channels are found in the presynaptic terminals of neurones, where they have a key role in synaptic transmission. They are also found in the somata of many cells, in dendrites and along a few axons. In no cell is the actual distribution of these channels known in detail, because there are no known toxins or other agents suitable for labelling calcium channels, and the current through these channels is usually too small to be quantified with extracellular electrodes. However, several experiments have suggested that the density of the channels is less in the axon than in the cell body or terminal region. Here we have used the indicator dye Arsenazo III in conjunction with an array of photodetectors to examine the spatial influx of calcium in the presynaptic terminal region of the giant barnacle, Balanus nubilus. In these cells, calcium entry occurs in a restricted region less than 50 micron in length, which corresponds closely to the region of synaptic contact with second-order cells. Outside this area the magnitude of calcium entry is reduced at least 50-fold. With reasonable assumptions it follows that the calcium channel density is equally localized. In addition, we demonstrate that these cells have a calcium-activated potassium conductance. Since calcium entry is restricted to the synaptic zone, this conductance must be effective only in this region.     

The open pore conformation of potassium channels

Living cells regulate the activity of their ion channels through a process known as gating. To open the pore, protein conformational changes must occur within a channel's membrane-spanning ion pathway. KcsA and MthK, closed and opened K(+) channels, respectively, reveal how such gating transitions occur. Pore-lining 'inner' helices contain a 'gating hinge' that bends by approximately 30 degrees. In a straight conformation four inner helices form a bundle, closing the pore near its intracellular surface. In a bent configuration the inner helices splay open creating a wide (12 A) entryway. Amino-acid sequence conservation suggests a common structural basis for gating in a wide range of K(+) channels, both ligand- and voltage-gated. The open conformation favours high conduction by compressing the membrane field to the selectivity filter, and also permits large organic cations and inactivation peptides to enter the pore from the intracellular solution.     

The voltage-gated potassium channels and their relatives

The voltage-gated potassium channels are the prototypical members of a family of membrane signalling proteins. These protein-based machines have pores that pass millions of ions per second across the membrane with astonishing selectivity, and their gates snap open and shut in milliseconds as they sense changes in voltage or ligand concentration. The architectural modules and functional components of these sophisticated signalling molecules are becoming clear, but some important links remain to be elucidated.     

Voltage-dependent calcium and potassium channels in retinal glial cells

Glial cells, which outnumber neurones in the central nervous system, have traditionally been considered to be electrically inexcitable and to play only a passive role in the electrical activity of the brain. Recent reports have demonstrated, however, that certain glial cells, when maintained in primary culture, possess voltage-dependent ion channels. It remains to be demonstrated whether these channels are also present in glial cells in vivo. I show here that Müller cells, the principal glial cells of the vertebrate retina, can generate 'Ca2+ spikes' in freshly excised slices of retinal tissue. In addition, voltage-clamp studies of enzymatically dissociated Müller cells demonstrate the presence of four types of voltage-dependent ion channels: a Ca2+ channel, a Ca2+-activated K+ channel, a fast-inactivating (type A) K+ channel and an inward-rectifying K+ channel. Currents generated by these voltage-dependent channels may enhance the ability of Müller cells to regulate extracellular K+ levels in the retina and may be involved in the generation of the electroretinogram.     

Chloride and potassium channels in cystic fibrosis airway epithelia

Cystic fibrosis, the most common lethal genetic disease in Caucasians, is characterized by a decreased permeability in sweat gland duct and airway epithelia. In sweat duct epithelium, a decreased Cl- permeability accounts for the abnormally increased salt content of sweat. In airway epithelia a decreased Cl- permeability, and possibly increased sodium absorption, may account for the abnormal respiratory tract fluid. The Cl- impermeability has been localized to the apical membrane of cystic fibrosis airway epithelial cells. The finding that hormonally regulated Cl- channels make the apical membrane Cl- permeable in normal airway epithelial cells suggested abnormal Cl- channel function in cystic fibrosis. Here we report that excised, cell-free patches of membrane from cystic fibrosis epithelial cells contain Cl- channels that have the same conductive properties as Cl- channels from normal cells. However, Cl- channels from cystic fibrosis cells did not open when they were attached to the cell. These findings suggest defective regulation of Cl- channels in cystic fibrosis epithelia; to begin to address this issue, we performed two studies. First, we found that isoprenaline, which stimulates Cl- secretion, increases cellular levels of cyclic AMP in a similar manner in cystic fibrosis and non-cystic fibrosis epithelial cells. Second, we show that adrenergic agonists open calcium-activated potassium channels, indirectly suggesting that calcium-dependent stimulus-response coupling is intact in cystic fibrosis. These data suggest defective regulation of Cl- channels at a site distal to cAMP accumulation.     

Cell-free expression of functional Shaker potassium channels.

The functional activity of ion channels and other membrane proteins requires that the proteins be correctly assembled in a transmembrane configuration. Thus, the functional expression of ion channels, neurotransmitter receptors and complex membrane-limited signalling mechanisms from complementary DNA has required the injection of messenger RNA or transfection of DNA into Xenopus oocytes or other target cells that are capable of processing newly translated protein into the surface membrane. These approaches, combined with voltage-clamp analysis of ion channel currents, have been especially powerful in the identification of structure-function relationships in ion channels. But oocytes express endogenous ion channels, neurotransmitter receptors and receptor-channel subunits, complicating the interpretation of results in mRNA-injected eggs. Furthermore, it is difficult to control experimentally the membrane lipids and post-translational modifications that underlie the regulation and modulation of ion channels in intact cells. A cell-free system for ion channel expression is ideal for good experimental control of protein expression and modulatory processes. Here we combine cell-free protein translation, microsomal membrane processing of nascent channel proteins, and reconstitution of newly synthesized ion channels into planar lipid bilayers to synthesize, glycosylate, process into membranes, and record in vitro the activity of functional Shaker potassium channels.     

Voltage-dependent ATP-sensitive potassium channels of skeletal muscle membrane

It has been known for some years that skeletal muscle develops a high potassium permeability in conditions that produce rigor, where ATP concentrations are low and intracellular Ca2+ is high. It has seemed natural to attribute this high permeability to K channels that are opened by internal Ca2+, especially as the presence of such channels has been demonstrated in myotubes and in the transverse tubular membrane system of adult skeletal muscle. However, as we show here, the surface membrane of frog muscle contains potassium channels that open at low internal concentrations of ATP (less than 2 mM). ATP induces closing of these channels without being split, apparently holding the channels in one of a number of closed states. The channels have at least two open states whose dwell times are voltage-dependent. Surprisingly, we find that these may be the most common K channels of the surface membrane of skeletal muscle.     

Expression of functional potassium channels from Shaker cDNA in Xenopus oocytes

The Shaker gene of Drosophila melanogaster encodes a potassium-selective ion channel, the 'A' channel, or one of its subunits. A single Shaker messenger RNA species suffices to direct the synthesis of functional A channels in Xenopus oocytes. Physiological characteristics of the A currents induced by two different mRNA species are compared.     

Ion conduction pore is conserved among potassium channels.

Potassium channels, a group of specialized membrane proteins, enable K+ ions to flow selectively across cell membranes. Transmembrane K+ currents underlie electrical signalling in neurons and other excitable cells. The atomic structure of a bacterial K+ channel pore has been solved by means of X-ray crystallography. To the extent that the prokaryotic pore is representative of other K+ channels, this landmark achievement has profound implications for our general understanding of K+ channels. But serious doubts have been raised concerning whether the prokaryotic K+ channel pore does actually represent those of eukaryotes. Here we have addressed this fundamental issue by substituting the prokaryotic pore into eukaryotic voltage-gated and inward-rectifier K+ channels. The resulting chimaeras retain the respective functional hallmarks of the eukaryotic channels, which indicates that the ion conduction pore is indeed conserved among K+ channels.     

Evidence for the formation of heteromultimeric potassium channels in Xenopus oocytes

Potassium channels show a wide range of functional diversity. Nerve cells typically express a number of K+ channels that differ in their kinetics, single-channel conductance, pharmacology, and sensitivity to voltage and second messengers. The cloning of the Shaker gene in Drosophila, and of related genes, has revealed that the encoded K+ channel polypeptides resemble one of the four internally homologous domains of the alpha-subunits of Na+ channels and Ca2+ channels, indicating that K+ channels may form by the co-assembly of several polypeptides. In this report we provide evidence that the Shaker A-type K+ channels expressed in Xenopus oocytes contain several Shaker polypeptides, that heteromultimeric channels may form through assembly of different channel polypeptides, that the kinetics or pharmacology of some heteromultimeric channels differ from those of homomultimeric channels, and that channel polypeptides from the fruit fly can co-assemble with homologous polypeptides from the rat. We suggest that heteromultimer formation may increase K+ channel diversity beyond even the level expected from the large number of K+ channel genes and alternative splicing products.     

Modulation of A-type potassium channels by a family of calcium sensors

In the brain and heart, rapidly inactivating (A-type) voltage-gated potassium (Kv) currents operate at subthreshold membrane potentials to control the excitability of neurons and cardiac myocytes. Although pore-forming alpha-subunits of the Kv4, or Shal-related, channel family form A-type currents in heterologous cells, these differ significantly from native A-type currents. Here we describe three Kv channel-interacting proteins (KChIPs) that bind to the cytoplasmic amino termini of Kv4 alpha-subunits. We find that expression of KChIP and Kv4 together reconstitutes several features of native A-type currents by modulating the density, inactivation kinetics and rate of recovery from inactivation of Kv4 channels in heterologous cells. All three KChIPs co-localize and co-immunoprecipitate with brain Kv4 alpha-subunits, and are thus integral components of native Kv4 channel complexes. The KChIPs have four EF-hand-like domains and bind calcium ions. As the activity and density of neuronal A-type currents tightly control responses to excitatory synaptic inputs, these KChIPs may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium.