Depletion of intracellular calcium stores activates a calcium current in mast cells.

In many cell types, receptor-mediated Ca2+ release from internal stores is followed by Ca2+ influx across the plasma membrane. The sustained entry of Ca2+ is thought to result partly from the depletion of intracellular Ca2+ pools. Most investigations have characterized Ca2+ influx indirectly by measuring Ca(2+)-activated currents or using Fura-2 quenching by Mn2+, which in some cells enters the cells by the same influx pathway. But only a few studies have investigated this Ca2+ entry pathway more directly. We have combined patch-clamp and Fura-2 measurements to monitor membrane currents in mast cells under conditions where intracellular Ca2+ stores were emptied by either inositol 1,4,5-trisphosphate, ionomycin, or excess of the Ca2+ chelator EGTA. The depletion of Ca2+ pools by these independent mechanisms commonly induced activation of a sustained calcium inward current that was highly selective for Ca2+ ions over Ba2+, Sr2+ and Mn2+. This Ca2+ current, which we term ICRAC (calcium release-activated calcium), is not voltage-activated and shows a characteristic inward rectification. It may be the mechanism by which electrically nonexcitable cells maintain raised intracellular Ca2+ concentrations and replenish their empty Ca2+ stores after receptor stimulation.     

Chloride conductance regulated by cyclic AMP-dependent protein kinase in cardiac myocytes

In heart cells, cyclic AMP-dependent protein kinase (PKA) regulates calcium- and potassium-ion current by phosphorylating the ion channels or closely associated regulatory proteins. We report here that isoprenaline induced large chloride-ion currents in voltage-clamped, internally-dialysed myocytes from guinea-pig ventricles. The Cl- current could be activated by intracellular dialysis with cAMP or the catalytic subunit of PKA, indicating regulation by phosphorylation. In approximately symmetrical solutions of high Cl- concentration, the macroscopic cardiac Cl- current showed little rectification, unlike the single-channel current in PKA-regulated Cl- channels of airway epithelial cells. But, like epithelial Cl- -channel currents, the cardiac Cl- current was sensitive to the distilbene,4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS). In the absence of kinase activation, cardiac sarcolemmal Cl- conductance was negligible. During beta-adrenergic stimulation of the heart, this novel Cl- conductance should accelerate action-potential repolarization and so protect impulse propagation in the face of the possibly arrhythmogenic increases in heart rate and in calcium entry into the cells.     

Uncoupling of cardiac muscarinic and beta-adrenergic receptors from ion channels by a guanine nucleotide analogue

Guanine nucleotide binding proteins, interchangeably called N or G proteins, seem to be the primary signal-transducing components of various agonist-induced cell membrane functions. In the heart, G proteins have been implicated in beta-adrenergic modulation of the slow inward Ca2+ current. We have investigated the role of G proteins in muscarinic activation of an inwardly rectifying, acetylcholine (ACh)-induced K+ current (IACh), and beta-adrenergic activation of an (isoprenaline)-induced Ca2+ current (Isi). Here we report that intracellular application of the non-hydrolysable GTP analogue 5'-guanylylimidodiphosphate (GppNHp) brought about an agonist-induced, antagonist-resistant, persistent activation of IACh and Isi. This functional uncoupling of channel from receptor suggests that the muscarinic receptor and the IACh channel are separate molecular structures. Membrane conductance responses to sequential activation of muscarinic and beta-adrenergic receptors demonstrate that in contrast to the muscarinic inhibition of Isi, muscarinic stimulation of IACh is mediated by a G protein via a pathway that does not involve adenylate cyclase. Taken together, the results support the notion that agonist is required to induce GppNHp binding and/or activation of the G proteins. Once triggered by agonist, the control system remains maximally activated, thereby transforming the cell so that it no longer responds to subsequent homologous receptor-mediated signals.     

Low intracellular pH and chemical agents slow inactivation gating in sodium channels of muscle

Excitation of nerve or muscle requires an orderly opening and closing of molecular pores, the ionic channels, in the plasma membrane. During the action potential, Na channels are opened (activated) by the advancing wave of depolarisation, contributing a pulse of inward sodium current, and then are closed again (inactivated) by the continued depolarisation. As one approach both to obtaining molecular information on the Na channel and towards further defining the recently discovered kinetic interactions of the inactivation and activation gating steps, we have surveyed here the effects of chemical agents reported to slow or prevent Na channel inactivation. We find that many of the agents studied by others on invertebrate giant axons or vertebrate nerve act on our frog skeletal muscle preparation. In addition, we have discovered that simply lowering the intracellular pH nearly eliminates inactivation. The activation mechanism seems to resist modification.     

Beta-adrenergic modulation of calcium channels in frog ventricular heart cells

Adrenergic modulation of calcium channels profoundly influences cardiac function, and has served as a prime example of neurohormonal regulation of voltage-gated ion channels. Channel modulation and increased Ca influx are mediated by elevation of intracellular cyclic AMP and protein phosphorylation. The molecular mechanism of the augmented membrane Ca conductance has attracted considerable interest. An increase in the density of functional channels has often been proposed, but there has previously been no direct evidence. Single-channel recordings show that isoprenaline or 8-bromocyclic AMP increase the proportion of time individual channels spend open by prolonging openings and shortening the closed periods between openings. To look for an additional contribution of changes in the number of functional channels, we applied ensemble fluctuation analysis to whole-cell recordings of cardiac Ca channel activity. Here we present evidence that in frog ventricular heart cells beta-adrenergic stimulation increases NF, the average number of functional Ca channels per cell. We also find that isoprenaline slows the time course of both activation and inactivation, and that the enhancement of peak current decreases gradually with greater membrane depolarization.     

Role for microsomal Ca storage in mammalian neurones?

Alterations in the intracellular concentration of calcium ions [( Ca2+]i) are increasingly being found to be associated with regulatory functions in cells of all kinds. In muscle, an elevation of [Ca2+]i is the final link in excitation-contraction coupling while at nerve endings and in secretory cells, similar rises in [Ca2+]i are thought to mediate exocytosis. The discovery of calcium-activated ion channels indicated a role for intracellular calcium in the regulation of membrane excitability. Calcium transients associated with either intracellular release or the inward movement of Ca2+ across the membrane have been recorded in molluscan neurons and more recently in neurones of bullfrog sympathetic ganglia. Here, we report the first recordings of calcium transients in single mammalian neurones. In these experiments we have found that the methylxanthine, caffeine, causes the release of calcium from a labile intracellular store which can be refilled by Ca2+ entering the cell during action potentials.     

Mechanism of calcium channel blockade by verapamil, D600, diltiazem and nitrendipine in single dialysed heart cells

Organic inhibitors of calcium influx prevent outward as well as inward current through cardiac calcium channels but do not slow current activation. Although block is antagonized by raising external calcium or barium concentrations, the competitive effect of permeant cations does not occur at the same cation binding site at which inorganic blockers act. Organic drugs show varying degrees of use-dependent block, due in part to blockade of open channels. Nitrendipine blockade of calcium currents requires doses greater than 100-fold higher than expected from radioligand binding to isolated membranes.     

Developmental acquisition of Ca2+-sensitivity by K+ channels in spinal neurones

A developmental change in the ionic basis of the inward current of action potentials has been observed in many excitable cells. In cultured spinal neurones of Xenopus, the timing of the development of the action parallels that seen in vivo. In vitro, as in vivo, neurones initially produce action potentials of long duration which are principally Ca-dependent; after 1 day of development the impulse is brief and primarily Na-dependent. At both ages, however, both inward components are present and the mechanism underlying shortening of the action potential is unknown. One possibility is that the outward currents change during development. Using the patch-clamp technique, we have recorded single K+-channel currents in membrane patches isolated from the cell bodies of cultured embryonic neurones. The unitary conductance of one class of K+ channels was approximately 155 pS and depolarization increased the probability of a channel being open. Neither conductance nor voltage dependence seemed to change with time in culture; in contrast, the Ca2+-sensitivity of this K+ channel increased. In younger neurones, Ca2+-sensitivity was greatly reduced or absent, whereas in more mature neurones, the activity of this channel was Ca-dependent. Such a change could account for the shortening of the action potential duration by increasing the relative contribution of outward currents.     

Potentiation of NMDA receptor currents by arachidonic acid.

Arachidonic acid is released by phospholipase A2 when activation of N-methyl-D-aspartate (NMDA) receptors by neurotransmitter glutamate raises the calcium concentration in neurons, for example during the initiation of long-term potentiation and during brain anoxia. Here we investigate the effect of arachidonic acid on glutamate-gated ion channels by whole-cell clamping isolated cerebellar granule cells. Arachidonic acid potentiates, and makes more transient, the current through NMDA receptor channels, and slightly reduces the current through non-NMDA receptor channels. Potentiation of the NMDA receptor current results from an increase in channel open probability, with no change in open channel current. We observe potentiation even with saturating levels of agonist at the glutamate- and glycine-binding sites on these channels; it does not result from conversion of arachidonic acid to lipoxygenase or cyclooxygenase derivatives, or from activation of protein kinase C. Arachidonic acid may act by binding to a site on the NMDA receptor, or by modifying the receptor's lipid environment. Our results suggest that arachidonic acid released by activation of NMDA (or other) receptors will potentiate NMDA receptor currents, and thus amplify increases in intracellular calcium concentration caused by glutamate. This may explain why inhibition of phospholipase A2 blocks the induction of long-term potentiation.     

Activation of a calcium-dependent photoprotein by chemical signalling through gap junctions

In the hydrozoan coelenterate Obelia geniculata, epithelial cell action potentials trigger light emission from photocyte effector cells containing obelin, an endogenous calcium-activated photoprotein. As this luminescence is blocked by the removal of extracellular calcium it seemed likely that calcium entry via voltage-gated channels in the photocyte membrane would account for the light emission. However, no inward calcium current was detected in whole cell recordings from dissociated photocytes and depolarization of isolated photocytes produced no luminescence. In contrast, a voltage-dependent calcium current was recorded from non-luminescent support cells, and activation of this current triggered luminescence in an adjacent photocyte. Surprisingly, light emission was abolished when the gap junctions between the photocyte and support cell were blocked. We conclude that calcium entry into support cells leads to light emission from neighbouring photocytes via chemical signalling through intercellular gap junctions.     

Ohmic conductance through the inwardly rectifying K channel and blocking by internal Mg2+

The inwardly rectifying K channel provides the resting K conductance in a variety of cells. This channel acts as a valve or diode, permitting entry of K+ under hyperpolarization, but not its exit under depolarization. This behaviour, termed inward rectification, permits long depolarizing responses which are of physiological significance for the pumping function of the heart and for fertilization of egg cells. Little is known about the outward currents through the inwardly rectifying K channel, despite their great physiological importance, and the mechanism of inward rectification itself is unknown. We have used improved patch clamp techniques to control the intracellular media, and have recorded the outward whole-cell and single-channel currents. We report here that the channel conductance is ohmic and that the well-known inward rectification of the resting K conductance is caused by rapid closure of the channel accompanied by a voltage-dependent block by intracellular Mg2+ ions at physiological concentrations.     

Elevation of intracellular calcium reduces voltage-dependent potassium conductance in human T cells

Both voltage-activated potassium channels and the concentration of free intracellular calcium have been implicated in the activation of T lymphocytes. Using the patch-clamp technique, we now show an unexpected relationship between the level of intracellular calcium [Ca]i in human lymphocytes and the amplitude of a voltage-dependent current: the elevation of [Ca]i decreases the potassium conductance. This is in contrast to other systems where [Ca]i activates K+ channels. Our results suggest that the level of intracellular calcium regulates the effective number of K+ channels capable of being activated.     

Acetylcholine activates an inward current in single mammalian smooth muscle cells

Acetylcholine, the major excitatory neurotransmitter to the smooth muscle of mammalian intestine, is known to depolarize smooth muscle cells with an apparent increase in membrane conductance. However, the ionic mechanisms that are triggered by muscarinic receptor activation and underlie this response are poorly understood, due in part to the technical problems associated with the electrophysiological study of smooth muscle. The muscarinic action of acetylcholine in certain neurones has been shown to involve the switching off of a resting K+ current (M-current) and a similar mechanism has recently also been identified in smooth muscle of amphibian stomach. We have now applied the patch-clamp technique to single smooth muscle cells of rabbit jejunum and find that muscarinic receptor activation switches on a nonselective, voltage-sensitive inward current. In addition, acetylcholine activates and then suppresses spontaneous K+ current transients, which are probably triggered by rises in intracellular Ca2+ in these cells.     

Activation of Ca2+ entry into acinar cells by a non-phosphorylatable inositol trisphosphate.

In many cell types, receptor activation of phosphoinositidase C results in an initial release of intracellular Ca2+ stores followed by sustained Ca2+ entry across the plasma membrane. Inositol 1,4,5-trisphosphate is the mediator of the initial Ca2+ release, although its role in the mechanism underlying Ca2+ entry remains controversial. We have now used two techniques to introduce inositol phosphates into mouse lacrimal acinar cells and measure their effects on Ca2+ entry: microinjection into cells loaded with Fura-2, a fluorescent dye which allows the measurement of intracellular free calcium concentration by microspectrofluorimetry, and perfusion of patch clamp pipettes in the whole-cell configuration while monitoring the activity of Ca(2+)-activated K+ channels as an indicator of intracellular Ca2+. We report here that inositol 1,4,5-trisphosphate serves as a signal that is both necessary and sufficient for receptor activation of Ca2+ entry across the plasma membrane in these cells.     

pH-sensitive activation of the intracellular-pH regulation system in squid axons by ATP-gamma-S

The regulation of intracellular pH (pHi) is essential for normal cell function, and controlled changes in pHi may play a central role in cell activation. Sodium-dependent Cl-HCO3 exchange is the dominant mechanism of pHi regulation in the invertebrate cells examined, and also occurs in mammalian cells. The transporter extrudes acid from the cell by exchanging extracellular Na+ and HCO3- (ref. 9) (or a related species) for intracellular Cl- (refs 3, 4). It is blocked by the stilbene derivatives DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulphonate, ref. 10) and SITS (4-acetamido-4'-isothiocyano-stilbene-2,2'-disulphonate, ref. 3), and has a stoichiometry of two intracellular H+ neutralized for each Na+ taken up and each Cl- extruded by the axon. Because the inwardly-directed Na+ concentration gradient is sufficiently large to energize both the HCO3- influx and Cl- efflux, this electroneutral exchanger could be a classic secondary active transporter, thermodynamically independent of ATP hydrolysis. However, at least in the squid axon, the exchanger has an absolute requirement for ATP (ref. 3). Thus, a major unresolved issue is whether this Na-dependent Cl-HCO3 exchanger stoichiometrically hydrolyses ATP (the pump hypothesis), or whether ATP activates the transporter by a mechanism such as phosphorylation or simple binding (the activation hypothesis). We have now explored the role of ATP in pHi regulation by dialysing axons with the ATP analogue ATP-gamma-S. In many systems, ATP-gamma-S is an acceptable substrate for protein kinases, whereas the resulting thiophosphorylated proteins are not as readily hydrolysed by phosphatases as are phosphorylated proteins. Our results rule out the pump hypothesis, and show that the basis of the axon's ATP requirement is the pH-dependent activation (by, for instance, phosphorylation or ATP binding) of the exchanger itself, or of an essential activator.     

Role of cyclic AMP in a serotonin-evoked slow inward current in snail neurones

One model of synaptic transmission suggests that transmitters modify postsynaptic permeability through the intermediary of cyclic AMP. Thus, serotonin (5-hydroxytryptamine) evokes in molluscan neurones a decrease in a voltage-dependent K+ conductance which in turn generates a slow inward current when studied in steady voltage-clamp conditions. The serotonin-induced increase of the plateau phase of the spike of an Aplysia sensory neurone can be mimicked by both intracellularly injected cyclic AMP and extracellularly applied phosphodiesterase inhibitors, suggesting that cyclic AMP mediates the effect. We have tested whether a similar mechanism could account for the serotonin slow inward current in identified snail neurones and have found that the intracellular injection of cyclic AMP, but not of cyclic GMP or 5'-AMP, evokes a slow inward current showing similar voltage dependence, inversion potential and ionic properties to the serotonin slow inward current. Phosphodiesterase inhibitors at low concentrations (1-20 microM) potentiate the serotonin slow inward current and at higher concentrations evoke by themselves an inward current, partially or totally occluding the serotonin and cyclic AMP currents. Finally, we have found that in homogenates of pooled identified snail neurones serotonin stimulates the adenylate cyclase, increasing its activity by 50-100%.     

Regulation of calcium influx by second messengers in rat mast cells

Biphasic increases in the free intracellular calcium concentration, consisting of a large initial transient followed by a sustained elevation, are frequently observed in non-excitable cells following stimulation. In rat peritoneal mast cells a cAMP- and Ca-activated chloride current can interact with IP3-dependent calcium influx to provide the sustained elevation of intracellular Ca concentration following transient IP3-induced release of calcium from intracellular stores. This novel combination of second messenger systems provides a flexible means to modulate calcium-dependent processes such as exocytosis.     

Protein kinase C activation induces conductance changes in Hermissenda photoreceptors like those seen in associative learning

Phosphorylation of ion channels has been suggested as one molecular mechanism responsible for learning-produced long-term changes in neuronal excitability. Persistent training-produced changes in two distinct K+ currents (IA (ref. 2), IK-Ca (refs 3,4)) and a voltage-dependent calcium current (ICa; refs 3,4) have previously been shown to occur in type B photoreceptors of Hermissenda, as a result of associative learning. But the identity of the phosphorylation pathway(s) responsible for these changes has not as yet been determined. Injections of cyclic AMP-dependent protein kinase reduce a K+ current (IK) in B cells which is different from those changed by training, but fails to reduce IA and IK-Ca. Phosphorylase b kinase (an exogenous calcium/calmodulin-dependent kinase) reduces IA, but whether IK-Ca and ICa are changed in the manner of associative training is not yet known. Another protein kinase present in high concentrations in both mammalian brain and molluscan nervous systems is protein kinase C, which is both calcium- and phospholipid-sensitive. We now present evidence that activation of protein kinase C by the tumour promoter phorbol ester (PDB) and intracellular injection of the enzyme induce conductance changes similar to those caused by associative training in Hermissenda B cells (that is a reduction of IA and IK-Ca, and enhancement of ICa). These results represent the first direct demonstration that protein kinase C affects membrane K+ ion conductance mechanisms.     

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.     

Synaptic excitation produces a long-lasting rebound potentiation of inhibitory synaptic signals in cerebellar Purkinje cells.

Persistent changes in synaptic efficacy are thought to underlie the formation of learning and memory in the brain. High-frequency activation of an afferent excitatory fibre system can induce long-term potentiation, and conjunctive activation of two distinct excitatory synaptic inputs to the cerebellar Purkinje cells can lead to long-term depression of the synaptic activity of one of the inputs. Here we report a new form of neural plasticity in which activation of an excitatory synaptic input can induce a potentiation of inhibitory synaptic signals to the same cell. In cerebellar Purkinje cells stimulation of the excitatory climbing fibre synapses is followed by a long-lasting (up to 75 min) potentiation of gamma-aminobutyric acid A (GABAA) receptor-mediated inhibitory postsynaptic currents (i.p.s.cs), a phenomenon that we term rebound potentiation. Using whole-cell patch-clamp recordings in combination with fluorometric video imaging of intracellular calcium ion concentration, we find that a climbing fibre-induced transient increase in postsynaptic calcium concentration triggers the induction of rebound potentiation. Because the response of Purkinje cells to bath-applied exogenous GABA is also potentiated after climbing fibre-stimulation with a time course similar to that of the rebound potentiation of i.p.s.cs, we conclude that the potentiation is caused by a calcium-dependent upregulation of postsynaptic GABAA receptor function. We propose that rebound potentiation is a mechanism by which in vivo block of climbing fibre activity induces an increase in excitability in Purkinje cells. Moreover, rebound potentiation of i.p.s.cs is a cellular mechanism which, in addition to the long-term depression of parallel fibre synaptic activity, may have an important role for motor learning in the cerebellum.