Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle

Action potentials in many excitable cells are followed by a prolonged afterhyperpolarization that modulates repetitive firing. Although it is established that the afterhyperpolarization is produced by Ca-activated K+ currents, the basis of these currents is not known. The large conductance (250 pS) Ca-activated K+ channel (BK channel) is not a major contributor to the afterhyperpolarization in non-innervated skeletal muscle and some nerve cells, because apamin, a neurotoxic component of bee venom, abolishes the afterhyperpolarization but does not block BK channels, and 5 mM extracellular tetraethylammonium ion (TEA) blocks BK channels but does not reduce the afterhyperpolarization. We now report single-channel currents from small conductance (10-14 pS) Ca-activated K+ channels (SK channels) with the necessary properties to account for the afterhyperpolarization. SK channels are blocked by apamin but not by 5 mM external TEA (TEAo). They are also highly Ca-sensitive at the negative membrane potentials associated with the afterhyperpolarization.     

Somatostatin stimulates Ca(2+)-activated K+ channels through protein dephosphorylation.

The neuropeptide somatostatin inhibits secretion from electrically excitable cells in the pituitary, pancreas, gut and brain. In mammalian pituitary tumour cells somatostatin inhibits secretion through two distinct pertussis toxin-sensitive mechanisms. One involves inhibition of adenylyl cyclase, the other an unidentified cyclic AMP-independent mechanism that reduces Ca2+ influx by increasing membrane conductance to potassium. Here we demonstrate that the predominant electrophysiological effect of somatostatin on metabolically intact pituitary tumour cells is a large, sustained increase in the activity of the large-conductance Ca(2+)- and voltage-activated K+ channels (BK). This action of somatostatin does not involve direct effects of Ca2+, cAMP or G proteins on the channels. Our results indicate instead that somatostatin stimulates BK channel activity through protein dephosphorylation.     

Intracellular ATP directly blocks K+ channels in pancreatic B-cells

It is known that glucose-induced depolarization of pancreatic B-cells is due to reduced membrane K+-permeability and is coupled to an increase in the rate of glycolysis, but there has been no direct evidence linking specific metabolic processes or products to the closing of membrane K+ channels. During patch-clamp studies of proton inhibition of Ca2+-activated K+ channels [GK(Ca)] in B-cells, we identified a second K+-selective channel which is rapidly and reversibly inhibited by ATP applied to the cytoplasmic surface of the membrane. This channel is spontaneously active in excised patches and frequently coexists with GK(Ca) channels yet is insensitive to membrane potential and to intracellular free Ca2+ and pH. Blocking of the channel is ATP-specific and appears not to require metabolism of the ATP. This ATP-sensitive K+ channel [GK(ATP)] may be a link between metabolism and membrane K+-permeability in pancreatic B-cells.     

TRIC channels are essential for Ca2+ handling in intracellular stores

Cell signalling requires efficient Ca2+ mobilization from intracellular stores through Ca2+ release channels, as well as predicted counter-movement of ions across the sarcoplasmic/endoplasmic reticulum membrane to balance the transient negative potential generated by Ca2+ release. Ca2+ release channels were cloned more than 15 years ago, whereas the molecular identity of putative counter-ion channels remains unknown. Here we report two TRIC (trimeric intracellular cation) channel subtypes that are differentially expressed on intracellular stores in animal cell types. TRIC subtypes contain three proposed transmembrane segments, and form homo-trimers with a bullet-like structure. Electrophysiological measurements with purified TRIC preparations identify a monovalent cation-selective channel. In TRIC-knockout mice suffering embryonic cardiac failure, mutant cardiac myocytes show severe dysfunction in intracellular Ca2+ handling. The TRIC-deficient skeletal muscle sarcoplasmic reticulum shows reduced K+ permeability, as well as altered Ca2+ 'spark' signalling and voltage-induced Ca2+ release. Therefore, TRIC channels are likely to act as counter-ion channels that function in synchronization with Ca2+ release from intracellular stores.     

Quantification of Ca2+-activated K+ channels under hormonal control in pig pancreas acinar cells

Ca2+- and voltage-activated K+ channels are found in many electrically excitable cells and have an important role in regulating electrical activity. Recently, the large K+ channel has been found in the baso-lateral plasma membranes of salivary gland acinar cells, where it may be important in the regulation of salt transport. Using patch-clamp methods to record single-channel currents from excised fragments of baso-lateral acinar cell membranes in combination with current recordings from isolated single acinar cells and two- and three-cell clusters, we have now for the first time characterized the K+ channels quantitatively. In pig pancreatic acini there are 25-60 K+ channels per cell with a maximal single channel conductance of about 200 pS. We have quantified the relationship between internal ionized Ca2+ concentration [( Ca2+]i) membrane potential and open-state probability (p) of the K+ channel. By comparing curves obtained from excised patches relating membrane potential to p, at different levels of [Ca2+]i, with similar curves obtained from intact cells, [Ca2+]i in resting acinar cells was found to be between 10(-8) and 10(-7) M. In microelectrode experiments acetylcholine (ACh), gastrin-cholecystokinin (CCK) as well as bombesin peptides evoked Ca2+-dependent opening of the K+ conductance pathway, resulting in membrane hyperpolarization. The large K+ channel, which is under strict dual control by internal Ca2+ and voltage, may provide a crucial link between hormone-evoked increase in internal Ca2+ concentration and the resulting NaCl-rich fluid secretion.     

Functional analysis of an archaebacterial voltage-dependent K+ channel

All living organisms use ion channels to regulate the transport of ions across cellular membranes. Certain ion channels are classed as voltage-dependent because they have a voltage-sensing structure that induces their pores to open in response to changes in the cell membrane voltage. Until recently, the voltage-dependent K+, Ca2+ and Na+ channels were regarded as a unique development of eukaryotic cells, adapted to accomplish specialized electrical signalling, as exemplified in neurons. Here we present the functional characterization of a voltage-dependent K+ (K(V)) channel from a hyperthermophilic archaebacterium from an oceanic thermal vent. This channel possesses all the functional attributes of classical neuronal K(V) channels. The conservation of function reflects structural conservation in the voltage sensor as revealed by specific, high-affinity interactions with tarantula venom toxins, which evolved to inhibit eukaryotic K(V) channels.     

Voltage and Ca2+-activated K+ channel in baso-lateral acinar cell membranes of mammalian salivary glands

Nervous or hormonal stimulation of many exocrine glands evokes release of cellular K+ (ref. 1), as originally demonstrated in mammalian salivary glands2,3, and is associated with a marked increase in membrane conductance1,4,5. We now demonstrate directly, by using the patch-clamp technique6, the existence of a K+ channel with a large conductance localized in the baso-lateral plasma membranes of mouse and rat salivary gland acinar cells. The K+ channel has a conductance of approximately 250 pS in the presence of high K+ solutions on both sides of the membrane. Although mammalian exocrine glands are believed not to possess voltage-activated channels1,7, the probability of opening the salivary gland K+ channel was increased by membrane depolarization. The frequency of channel opening, particularly at higher membrane potentials, was increased markedly by elevating the internal ionized Ca2+ concentration, as previously shown for high-conductance K+ channels from cells of neural origin8-10. The Ca2+ and voltage-activated K+ channel explains the marked cellular K+ release that is characteristically observed when salivary glands are stimulated to secrete.     

Modulation of dihydropyridine-sensitive Ca2+ channels by glucose metabolism in mouse pancreatic beta-cells

Glucose stimulates insulin secretion from the pancreatic beta-cell by increasing the cytosolic calcium concentration. It is believed that this increment results mainly from Ca2+ influx through dihydropyridine-sensitive calcium channels because insulin secretion is abolished by dihydropyridine antagonists and is potentiated by dihydropyridine agonists. Glucose may influence Ca2+ influx through these channels in two ways: either by regulating the beta-cell membrane potential or by biochemical modulation of the channel itself. The former mechanism is well established. Glucose metabolism, by closing ATP-sensitive K+ channels, depolarizes the beta-cell membrane and initiates Ca2+-dependent electrical activity, with higher glucose concentrations further increasing Ca2+ influx by raising the frequency of action potentials. We show here that glucose metabolism also increases calcium influx directly, by modulating the activity of dihydropyridine-sensitive Ca2+ channels.     

Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin

Small-conductance Ca2+-activated K+ channels (SK channels) are independent of voltage and gated solely by intracellular Ca2+. These membrane channels are heteromeric complexes that comprise pore-forming alpha-subunits and the Ca2+-binding protein calmodulin (CaM). CaM binds to the SK channel through the CaM-binding domain (CaMBD), which is located in an intracellular region of the alpha-subunit immediately carboxy-terminal to the pore. Channel opening is triggered when Ca2+ binds the EF hands in the N-lobe of CaM. Here we report the 1.60 A crystal structure of the SK channel CaMBD/Ca2+/CaM complex. The CaMBD forms an elongated dimer with a CaM molecule bound at each end; each CaM wraps around three alpha-helices, two from one CaMBD subunit and one from the other. As only the CaM N-lobe has bound Ca2+, the structure provides a view of both calcium-dependent and -independent CaM/protein interactions. Together with biochemical data, the structure suggests a possible gating mechanism for the SK channel.     

Calcium activation of BK(Ca) potassium channels lacking the calcium bowl and RCK domains

In many physiological systems such as neurotransmitter release, smooth muscle relaxation and frequency tuning of auditory hair cells, large-conductance calcium-activated potassium (BK(Ca)) channels create a connection between calcium signalling pathways and membrane excitability. BK(Ca) channels are activated by voltage and by micromolar concentrations of intracellular calcium. Although it is possible to open BK(Ca) channels in the absence of calcium, calcium binding is essential for their activation under physiological conditions. In the presence of intracellular calcium, BK(Ca) channels open at more negative membrane potentials. Many experiments investigating the molecular mechanism of calcium activation of the BK(Ca) channel have focused on the large intracellular carboxy terminus, and much evidence supports the hypothesis that calcium-binding sites are located in this region of the channel. Here we show that BK(Ca) channels that lack the whole intracellular C terminus retain wild-type calcium sensitivity. These results show that the intracellular C terminus, including the 'calcium bowl' and the RCK domain, is not necessary for the calcium-activated opening of these channels.     

Mediation of cell volume regulation by Ca2+ influx through stretch-activated channels

Animal cells initially swell in hypotonic media by osmotic water equilibration, but their volume is subsequently regulated by a net loss of KCl and amino acids with concomitant loss of cell water. Mechanisms for regulating cell volume are important in allowing cells to adapt to variations in external tonicity and metabolic load. In red cells the KCl loss is mediated by electroneutral ion transport mechanisms. In contrast, conductive K+ and Cl- transport pathways are activated during regulatory volume decrease in several cell types including epithelia. The activation seems to be mediated by internal Ca2+, but the detailed mechanism is not known. In a leaky epithelium, the choroid plexus epithelium, we have found a cation-selective, Ca2+-permeable channel which opens with membrane stretch. The epithelium also contains a high density of the large (approximately 200 pS) type of Ca2+- voltage-activated K+ channel. Both channels are normally closed. I propose that in hypotonic media, the stretching of the cell membrane produced by the initial swelling causes influx of Ca2+ through the stretch-activated channels, which activates the neighbouring large K+ channels to produce increased K+ outflux with associated loss of cell water.     

Multiple intracellular signallings involved in regulation of on channels by GH releasing or inhibitory hormones in pituitary somatotropes

Influx of Ca2-via Ca2+ channels is the major step triggering exocytosis of pituitary somatotropes to release growth hormone (GH). Voltage-gated Ca2+ and K+ channels, the primary determinants of the influx of Ca2+ in somatotropes, are regulated by GH-releasing hornone (GHRH) and somatostatin (SRIF) through G protein-coupled signalling systems. Using whole-cell patch-clamp techniques, the changes of the Ca2+ and K+ currents in primary cultured somatotropes were recorded and signalling systems were studied using pharmacological reagents and intracellular dialysis of non-permeable molecules including antibodies and antisense oligonucleotides. GHRH increased both L-and T-types Ca2+ currents and decreased transient (I4) and delayed rectified (Ik) K+ currents. The increase in Ca2+ currents by GHRH was mediated by cAMP/protein kinase A system but the decrease in K+ currents required normal function of protein kinase C system. The GHRH-induced alteration of Ca2+ and K+ currents augments the influx of Ca2+ , leading to an increase in the [ Ca2+ ]I and the GH secretion. In contrary, a significant reduction in Ca2+ currents and increase in K currents were obtained in response to SRIF. The ion channel response to SRIF was demonstrated as a membrane delimited pathway and can be recorded by classic whole-cell configuration, Intracellular dialysis of anti-αi3 antibodies attenuated the increase in K + currents by SRIF whereas anti-αo antibodies blocked the reduction in the Ca2+ current by SRIF. Dialysis of antisense oligonucleotides specific for αo2 sub-units also attenuated the inhibition of SRIF on the Ca2+current. The Gi3 protein mediated the increase in K + currents and the Go2 protein mediated the reduction in the Ca2 +current by SRIF. The SRIF-induced alteration of Ca2 + and K + currents diminished the influx of Ca2+ , leading to a decrease in the [ Ca2+ ]I and the GH secretion. It is therefore concluded that multiple signalling systems are employed in the ion channel response to GHRH or SRIF in somatotropes, which leads to an increase or decrease in the GH secretion.     

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

The recent development of techniques for recording currents through single ionic channels has led to the identification of a K+-specific channel that is activated by cytoplasmic Ca2+. The channel has complex properties, being activated by depolarizing voltages and having a voltage-sensitivity that is modulated by cytoplasmic Ca2+ levels. The conduction behaviour of the channel is also unusual, its high ionic selectivity being displayed simultaneously with a very high unitary conductance. Very little is known about the biochemistry of this channel, largely due to the lack of a suitable ligand for use as a biochemical probe for the channel. We describe here a protein inhibitor of single Ca2+-activated K+ channels of mammalian skeletal muscle. This inhibitor, a minor component of the venom of the Israeli scorpion, Leiurus quinquestriatus, reversibly blocks the large Ca2+-activated K+ channel in a simple biomolecular reaction. We have partially purified the active component, a basic protein of relative molecular mass (Mr) approximately 7,000.     

X-ray structure of a voltage-dependent K+ channel

Voltage-dependent K+ channels are members of the family of voltage-dependent cation (K+, Na+ and Ca2+) channels that open and allow ion conduction in response to changes in cell membrane voltage. This form of gating underlies the generation of nerve and muscle action potentials, among other processes. Here we present the structure of KvAP, a voltage-dependent K+ channel from Aeropyrum pernix. We have determined a crystal structure of the full-length channel at a resolution of 3.2 A, and of the isolated voltage-sensor domain at 1.9 A, both in complex with monoclonal Fab fragments. The channel contains a central ion-conduction pore surrounded by voltage sensors, which form what we call 'voltage-sensor paddles'-hydrophobic, cationic, helix-turn-helix structures on the channel's outer perimeter. Flexible hinges suggest that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.     

Detection of K+ and Cl-channels from calf cardiac sarcolemma in planar lipid bilayer membranes

The ionic currents underlying the cardiac action potential are believed to be much more complex than those in nerve. During the cardiac action potential, various membrane channels control the flow of K+, Na+, Ca2+ and Cl- across the sarcolemma of cardiac muscle cells. Thus, it has become increasingly clear that a detailed knowledge of the mechanisms that activate (or inactivate) heart channels is required to understand cardiac excitability. We report here the use of planar lipid bilayer techniques to detect and characterize K+ and Cl- channels in purified heart sarcolemma membrane vesicles. We have identified four different types of channel on the basis of their selectivity, conductance and gating kinetics. We present in some detail the properties of a K+ channel and a Cl- channel. We have tentatively identified the K+ channel with the ix type of current found in Purkinje, myocardial ventricular and atrial fibres. The chloride channel might be related to the transient chloride current found in Purkinje fibres.     

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.     

Inositol trisphosphate-dependent periodic activation of a Ca(2+)-activated K+ conductance in glucose-stimulated pancreatic beta-cells.

Glucose-stimulated insulin secretion is associated with the appearance of electrical activity in the pancreatic beta-cell. At intermediate glucose concentrations, beta-cell electrical activity follows a characteristic pattern of slow oscillations in membrane potential on which bursts of action potentials are superimposed. The electrophysiological background of the bursting pattern remains unestablished. Activation of Ca(2+)-activated large-conductance K+ channels (KCa channel) has been implicated in this process but seems unlikely in view of recent evidence demonstrating that the beta-cell electrical activity is unaffected by the specific KCa channel blocker charybdotoxin. Another hypothesis postulates that the bursting arises as a consequence of two components of Ca(2+)-current inactivation. Here we show that activation of a novel Ca(2+)-dependent K+ current in glucose-stimulated beta-cells produces a transient membrane repolarization. This interrupts action potential firing so that action potentials appear in bursts. Spontaneous activity of this current was seen only rarely but could be induced by addition of compounds functionally related to hormones and neurotransmitters present in the intact pancreatic islet. K+ currents of the same type could be evoked by intracellular application of GTP, the effect of which was mediated by mobilization of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores. These observations suggest that oscillatory glucose-stimulated electrical activity, which is correlated with pulsatile release of insulin, results from the interaction between the beta-cell and intraislet hormones and neurotransmitters. Our data also provide evidence for a close interplay between ion channels in the plasma membrane and InsP3-induced mobilization of intracellular Ca2+ in an excitable cell.     

Reversible inactivation of K+ channels of Vicia stomatal guard cells following the photolysis of caged inositol 1,4,5-trisphosphate

RECENT investigations suggest that cytoplasmic D-myo-inositol 1,4,5-trisphosphate (InsP3) functions as a second messenger in plants, as in animals, coupling environmental and other stimuli to intracellular Ca2+ release. Cytoplasmic levels of InsP3 and the turnover of several probable precursors in plants are affected by physiological stimuli--including light, osmotic stress and the phytohormone indoleacetic acid--and InsP3 activates Ca2+ channels and Ca2+ flux across plant vacuolar and microsomal membranes. Complementary data also link changes in cytoplasmic free Ca2+ to several physiological responses, notably in guard cells which regulate gas exchange through the stomatal pores of higher plant leaves. Recent evidence indicates that guard cell K+ channels and, hence, K+ flux for stomatal movements may be controlled by cytoplasmic Ca2+. So far, however, direct evidence of a role for InsP3 in signalling in plants has remained elusive. Here we report that InsP3 released from an inactive, photolabile precursor, the P5-1-(2-nitrophenyl)ethyl ester of InsP3 (caged InsP3) reversibly inactivates K+ channels thought to mediate K+ uptake by guard cells from Vicia faba L. while simultaneously activating an apparently time-independent, inward current to depolarize the membrane potential and promote K+ efflux through a second class of K+ channels. The data are consistent with a transient rise in cytoplasmic free Ca2+ and demonstrate that intact guard cells are competent to use InsP3 in signal cascades controlling ion flux through K+ channels.     

A functional correlate for the dihydropyridine binding site in rat brain

Calcium channels, controlling the influx of extracellular Ca2+ and hence neurotransmitter release, exist in the brain. However, drugs classed as calcium antagonists and which inhibit Ca2+ entry through voltage-activated Ca2+ channels in heart and smooth muscle, seem not to affect any aspect of neuronal function in the brain at pharmacologically relevant concentrations. Yet the dihydropyridine calcium antagonists (for example, nitrendipine) bind stereospecifically with high affinity to a recognition site on brain-cell membranes thought to represent the Ca2+ channel and consequently, the physiological relevance of these sites has been questioned. However, activation of voltage-dependent Ca2+ channels can increase cytoplasmic Ca2+ and neurotransmitter release in neuronal tissue. We show here that Bay K8644, a dihydropyridine Ca2+-channel activator, can augment K+-stimulated release of serotonin from rat frontal cortex slices and that these effects can be antagonized by low concentrations of calcium antagonists. As 3H-dihydropyridine binding to cortical membrane preparations resembles the binding in heart and smooth muscle where there are good functional correlates we conclude that the dihydropyridine binding sites in the brain represent functional Ca2+ channels that can be unmasked under certain circumstances.     

The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels

The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O2-, precursors to bactericidal reactive oxygen species). Electrons move from intracellular NADPH, across a chain comprising FAD (flavin adenine dinucleotide) and two haems, to reduce extracellular O2 to O2-. NADPH oxidase is electrogenic, generating electron current (I(e)) that is measurable under voltage-clamp conditions. Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that I(e) is voltage-independent from -100 mV to >0 mV, but is steeply inhibited by further depolarization, and is abolished at about +190 mV. It was proposed that H+ efflux mediated by voltage-gated proton channels compensates I(e), because Zn2+ and Cd2+ inhibit both H+ currents and O2- production. Here we show that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce O2- in the presence of Zn2+ concentrations that inhibit O2- production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.