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.     

Voltage-dependent InsP3-insensitive calcium channels in membranes of pancreatic endoplasmic reticulum vesicles.

Stimulus-secretion coupling in exocrine glands involves Ca2+ release from intracellular stores. In endoplasmic reticulum vesicle preparations from rat exocrine pancreas, an inositol 1,4,5-trisphosphate(InsP3)-sensitive, as well as an InsP3-insensitive, Ca2+ pool has been characterized. But Ca2+ channels in the endoplasmic reticulum of rat exocrine pancreas have not been demonstrated at the level of single-channel current. We have now used the patch-clamp technique on endoplasmic reticulum vesicles fused by means of the dehydration-rehydration method. In excised patches, single Ba2(+)- and Ca2(+)-selective channels were recorded. The channel activity was markedly voltage-dependent. Caffeine increased channel open-state probability, whereas ruthenium red and Cd2+ blocked single-channel currents. Ryanodine, nifedipine and heparin had no effect on channel activity. The channel activity was not dependent on the free Ca2+ concentration, the presence of InsP3, or pH. We conclude that this calcium channel mediates Ca2+ release from an intracellular store through an InsP3-insensitive mechanism.     

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.     

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.     

Acetylcholine activation of single muscarinic K+ channels in isolated pacemaker cells of the mammalian heart

Acetylcholine (ACh) released on vagal stimulation reduces the heart rate by increasing K+ conductance of pacemaker cells in the sinoatrial (S-A) node. Fluctuation analysis of ACh-activated currents in pacemaker tissue showed this to be due to opening of a separate class of K+ channels gated by muscarinic ACh receptors (m-AChRs). On the other hand, it has been suggested that m-AChRs may simply regulate the current flow through inward rectifying resting K+ channels (gk1). We report here the measurement of ACh-activated single channel K+ currents and of resting K+ channel currents in isolated cells of the atrioventricular (A-V) and S-A node of rabbit heart. The results show that the ACh-dependent K+ conductance increase in nodal cells is mediated by K+ channels which are different in their gating and conductance properties from the inward rectifying resting K+ channels in atrial and ventricular cells. The resting K+ channels in nodal cells are, however, similar to those activated by ACh.     

STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane

As the sole Ca2+ entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx is important in Ca2+ signalling and many other cellular processes. A calcium-release-activated calcium (CRAC) channel in T lymphocytes is the best-characterized SOC influx channel and is essential to the immune response, sustained activity of CRAC channels being required for gene expression and proliferation. The molecular identity and the gating mechanism of SOC and CRAC channels have remained elusive. Previously we identified Stim and the mammalian homologue STIM1 as essential components of CRAC channel activation in Drosophila S2 cells and human T lymphocytes. Here we show that the expression of EF-hand mutants of Stim or STIM1 activates CRAC channels constitutively without changing Ca2+ store content. By immunofluorescence, EM localization and surface biotinylation we show that STIM1 migrates from endoplasmic-reticulum-like sites to the plasma membrane upon depletion of the Ca2+ store. We propose that STIM1 functions as the missing link between Ca2+ store depletion and SOC influx, serving as a Ca2+ sensor that translocates upon store depletion to the plasma membrane to activate CRAC channels.     

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.     

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.     

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.     

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.     

Endfeet of retinal glial cells have higher densities of ion channels that mediate K+ buffering

A major function of glial cells in the central nervous system is to buffer the extracellular potassium concentration, [K+]o. A local rise in [K+]o causes potassium ions to enter glial cells, which have membranes that are highly permeable to K+; potassium then leaves the glial cells at other locations where [K+]o has not risen. We report here the first study of the individual ion channels mediating potassium buffering by glial cells. The patch-clamp technique was employed to record single channel currents in Müller cells, the radial glia of the vertebrate retina. Those cells have 94% of their potassium conductance in an endfoot apposed to the vitreous humour, causing K+ released from active retinal neurones to be buffered preferentially to the vitreous. Recordings from patches of endfoot and cell body membrane show that a single type of inward-rectifying K+ channel mediates potassium buffering at both cell locations. The non-uniform density of K+ conductance is due to a non-uniform distribution of one type of K+ channel, rather than to the cell expressing high conductance channels at the endfoot and low conductance channels elsewhere on the cell.     

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.     

Ca2+ signalling between single L-type Ca2+ channels and ryanodine receptors in heart cells

Ca2+-induced Ca2+ release is a general mechanism that most cells use to amplify Ca2+ signals. In heart cells, this mechanism is operated between voltage-gated L-type Ca2+ channels (LCCs) in the plasma membrane and Ca2+ release channels, commonly known as ryanodine receptors, in the sarcoplasmic reticulum. The Ca2+ influx through LCCs traverses a cleft of roughly 12 nm formed by the cell surface and the sarcoplasmic reticulum membrane, and activates adjacent ryanodine receptors to release Ca2+ in the form of Ca2+ sparks. Here we determine the kinetics, fidelity and stoichiometry of coupling between LCCs and ryanodine receptors. We show that the local Ca2+ signal produced by a single opening of an LCC, named a 'Ca2+ sparklet', can trigger about 4-6 ryanodine receptors to generate a Ca2+ spark. The coupling between LCCs and ryanodine receptors is stochastic, as judged by the exponential distribution of the coupling latency. The fraction of sparklets that successfully triggers a spark is less than unity and declines in a use-dependent manner. This optical analysis of single-channel communication affords a powerful means for elucidating Ca2+-signalling mechanisms at the molecular level.     

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.     

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.     

Ca2+ channel modulation by 8-bromocyclic AMP in cultured heart cells

Modulation of ion channels is of increasing interest as it is an important step in the regulation of cellular functions. We have analysed the effect of 8-bromocyclic AMP on Ca2+ channels in cultured cardiac cells by the patch-clamp method and report here that there was a large increase in the probability of opening of the channels. On the basis of a recently proposed kinetic reaction scheme we suggest that cyclic AMP-dependent phosphorylation of Ca2+ channels primarily promotes the forward rate constants which lead to the open state of a Ca2+ channel during depolarization.     

The large-conductance Ca2+-activated K+ channel is essential for innate immunity

Neutrophil leukocytes have a pivotal function in innate immunity. Dogma dictates that the lethal blow is delivered to microbes by reactive oxygen species (ROS) and halogens, products of the NADPH oxidase, whose impairment causes immunodeficiency. However, recent evidence indicates that the microbes might be killed by proteases, activated by the oxidase through the generation of a hypertonic, K+-rich and alkaline environment in the phagocytic vacuole. Here we show that K+ crosses the membrane through large-conductance Ca2+-activated K+ (BK(Ca)) channels. Specific inhibitors of these channels, iberiotoxin and paxilline, blocked oxidase-induced 86Rb+ fluxes and alkalinization of the phagocytic vacuole, whereas NS1619, a BK(Ca) channel opener, enhanced both. Characteristic outwardly rectifying K+ currents, reversibly inhibited by iberiotoxin, were demonstrated in neutrophils and eosinophils and the expression of the alpha-subunit of the BK channel was confirmed by western blotting. The channels were opened by the combination of membrane depolarization and elevated Ca2+ concentration, both consequences of oxidase activity. Remarkably, microbial killing and digestion were abolished when the BK(Ca) channel was blocked, revealing an essential and unexpected function for this K+ channel in the microbicidal process.     

Three types of neuronal calcium channel with different calcium agonist sensitivity

How many types of calcium channels exist in neurones? This question is fundamental to understanding how calcium entry contributes to diverse neuronal functions such as transmitter release, neurite extension, spike initiation and rhythmic firing. There is considerable evidence for the presence of more than one type of Ca conductance in neurones and other cells. However, little is known about single-channel properties of diverse neuronal Ca channels, or their responsiveness to dihydropyridines, compounds widely used as labels in Ca channel purification. Here we report evidence for the coexistence of three types of Ca channel in sensory neurones of the chick dorsal root ganglion. In addition to a large conductance channel that contributes long-lasting current at strong depolarizations (L), and a relatively tiny conductance that underlies a transient current activated at weak depolarizations (T), we find a third type of unitary activity (N) that is neither T nor L. N-type Ca channels require strongly negative potentials for complete removal of inactivation (unlike L) and strong depolarizations for activation (unlike T). The dihydropyridine Ca agonist Bay K 8644 strongly increases the opening probability of L-, but not T- or N-type channels.     

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.     

The vacuolar Ca2+-activated channel TPC1 regulates germination and stomatal movement

Cytosolic free calcium ([Ca2+]cyt) is a ubiquitous signalling component in plant cells. Numerous stimuli trigger sustained or transient elevations of [Ca2+]cyt that evoke downstream stimulus-specific responses. Generation of [Ca2+]cyt signals is effected through stimulus-induced opening of Ca2+-permeable ion channels that catalyse a flux of Ca2+ into the cytosol from extracellular or intracellular stores. Many classes of Ca2+ current have been characterized electrophysiologically in plant membranes. However, the identity of the ion channels that underlie these currents has until now remained obscure. Here we show that the TPC1 ('two-pore channel 1') gene of Arabidopsis thaliana encodes a class of Ca2+-dependent Ca2+-release channel that is known from numerous electrophysiological studies as the slow vacuolar channel. Slow vacuolar channels are ubiquitous in plant vacuoles, where they form the dominant conductance at micromolar [Ca2+]cyt. We show that a tpc1 knockout mutant lacks functional slow vacuolar channel activity and is defective in both abscisic acid-induced repression of germination and in the response of stomata to extracellular calcium. These studies unequivocally demonstrate a critical role of intracellular Ca2+-release channels in the physiological processes of plants.