Regional specialization of retinal glial cell membrane

Neural activity generates increases in extracellular K+ concentration, [K+]0, which must be regulated in order to maintain normal brain function. Glial cells are thought to play an important part in this regulation through the process of K+ spatial buffering: K+-mediated current flow through glial cells redistributes extracellular K+ following localized [K+]0 increases. As is the case in other glia, the retinal Müller cell is permeable almost exclusively to K+ . Recent experiments have suggested that this K+ conductance may not be distributed uniformly over the cell surface. In the present study, two novel techniques have been used to assess the Müller cell K+ conductance distribution. The results demonstrate that 94% of all membrane conductance lies in the endfoot process of the cell. This strikingly asymmetric distribution has important consequences for theories concerning K+ buffering and should help to explain the generation of the electroretinogram.     

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.     

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.     

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.     

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.     

Extrusion of calcium from rod outer segments is driven by both sodium and potassium gradients

Calcium is transported across the surface membrane of both nerve and muscle by a Na+-dependent mechanism, usually termed the Na:Ca exchange. It is well established from experiments on rod outer segments that one net positive charge enters the cell for every Ca2+ ion extruded by the exchange, which is generally interpreted to imply an exchange stoichiometry of 3 Na+:1 Ca2+. We have measured the currents associated with the operation of the exchange in both forward and reversed modes in isolated rod outer segments and we find that the reversed mode, in which Ca2+ enters the cell in exchange for Na+, depends strongly on the presence of external K+. The ability of changes in external K+ concentration ([K+]o) to perturb the equilibrium level of [Ca2+]i indicates that K+ is co-transported with calcium. From an examination of the relative changes of [Ca2+]o, [Na+]o, [K+]o and membrane potential required to maintain the exchange at equilibrium, we conclude that the exchange stoichiometry is 4 Na+:1 Ca2+, 1 K+ and we propose that the exchange should be renamed the Na:Ca, K exchange. Harnessing the outward K+ gradient should allow the exchange to maintain a Ca2+ efflux down to levels of internal [Ca2+] that are considerably lower than would be possible with a 3 Na+:1 Ca2+ exchange.     

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.     

Heteromultimeric channels formed by rat brain potassium-channel proteins

An important step towards understanding the molecular basis of the functional diversity of voltage-gated K+ channels in the mammalian brain has been the discovery of a family of genes encoding rat brain K+ channel-forming (RCK) proteins. All species of these RCK proteins form homomultimeric voltage-gated K+ channels with distinct functional characteristics in Xenopus laevis oocytes following injection of the respective cRNAs. RCK-specific mRNAs are coexpressed in several regions of the brain, suggesting that RCK proteins also assemble into heteromultimeric K+ channels. In addition expression experiments with fractionated poly(A)+ mRNA have suggested that heteromultimeric K+ channels may occur in mammalian brain. We report here that heteromultimeric K+ channels composed of two different RCK proteins (RCK1 and RCK4) assemble after cotransfection of HeLa cells with the corresponding cDNAs and after coinjection of the corresponding cRNAs into Xenopus oocytes. The heteromultimeric RCK1, 4 channel mediates a transient potassium outward current, similar to the RCK4 channel but inactivates more slowly, has a larger conductance and is more sensitive to block by dendrotoxin and tetraethylammonium chloride.     

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.     

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.     

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 glial cell glutamate uptake carrier countertransports pH-changing anions.

Uptake into glial cells helps to terminate glutamate's neurotransmitter action and to keep its extracellular concentration, [Glu]o, below neurotoxic levels. The accumulative power of the uptake carrier stems from its transport of inorganic ions such as sodium (into the cell) and potassium (out of the cell). There is controversy over whether the carrier also transports a proton (or pH-changing anion). Here we show that the carrier generates an alkalinization outside and an acidification inside glial cells, and transports anions out of the cells, suggesting that there is a carrier cycle in which two Na+ accompany each glutamate anion into the cell, while one K+ and one OH- (or HCO3-) are transported out. This stoichiometry predicts a minimum [Glu]o of 0.6 microM normally (tonically activating presynaptic autoreceptors and post-synaptic NMDA receptors), and 370 microM during brain anoxia (high enough to kill neurons). Transport of OH-/HCO3- on the uptake carrier generates significant pH changes, and may provide a mechanism for neuron-glial interaction.     

Putative receptor for the cytoplasmic inactivation gate in the Shaker K+ channel

Inactivation of ion channels is important in the control of membrane excitability. For example, delayed-rectifier K+ channels, which regulate action potential repolarization, are inactivated only slowly, whereas A-type K+ channels, which affect action potential duration and firing frequency, have both fast and slow inactivation. Fast inactivation of Na+ and K+ channels may result from the blocking of the permeation pathway by a positively charged cytoplasmic gate such as the one encoded by the first 20 amino acids of the Shaker B (ShB) K+ channel. We report here that mutation of five highly conserved residues between the proposed membrane-spanning segments S4 and S5 (also termed H4) of ShB affects the stability of the inactivated state and alters channel conductance. One such mutation stabilizes the inactivated state of ShB as well as the inactivated state induced in the delayed-rectifier type K+ channel drk1 by the cytoplasmic application of the ShB N-terminal peptide. The S4-S5 loop, therefore, probably forms part of a receptor for the inactivation gate and lies near the channel's permeation pathway.     

Ionic basis of membrane potential in outer hair cells of guinea pig cochlea

Mammalian hearing involves features not found in other species, for example, the separation of sound frequencies depends on an active control of the cochlear mechanics. The force-generating component in the cochlea is likely to be the outer hair cell (OHC), one of the two types of sensory cell through which current is gated by mechano-electrical transducer channels sited on the apical surface. Outer hair cells isolated in vitro have been shown to be motile and capable of generating forces at acoustic frequencies. The OHC membrane is not, however, electrically tuned, as found in lower vertebrates. Here we describe how the OHC resting potential is determined by a Ca2+-activated K+ conductance at the base of the cell. Two channel types with unitary sizes of 240 and 45 pS underlie this Ca2+-activated K+ conductance and we suggest that their activity is determined by a Ca2+ influx through the apical transducer channel, as demonstrated in other hair cells. This coupled system simultaneously explains the large OHC resting potentials observed in vivo and indicates how the current gated by the transducer may be maximized to generate the forces required in cochlear micromechanics.     

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.     

Single-channel and whole-cell recordings of mitogen-regulated inward currents in human cloned helper T lymphocytes

Cytoplasmic free Ca2+ [( Ca2+]i) appears to be an important signal for DNA synthesis in early stages of lymphocyte activation. In spite of many experimental studies which employ fluorescent Ca2+ indicator dye to demonstrate an early increase of [Ca2+]i in T-lymphocytes after stimulation with lectins, specific antigens, and monoclonal antibodies to T-lymphocyte receptors, the mechanism responsible for the rise of [Ca2+]i is unknown. We have used the extracellular patch clamp technique to investigate this mechanism. Unitary inward currents, mediated by Ca2+ or Ba2+, were recorded in the membrane of T-lymphocytes. The inward current channel was characterized by a conductance of 7 pS and extrapolated reversal potential (Erev) 110 mV positive to resting potential (Vr). While gating kinetic parameters were not affected by membrane potential changes, the probability of channel opening markedly increased upon activation of the T-lymphocyte by the mitogenic lectin, phytohaemagglutinin (PHA). PHA also evoked a cadmium-sensitive, inward Ba2+ current on whole-cell clamp. We suggest that this mitogen-regulated channel introduces Ca2+ into the cytoplasm upon activation and represents a new class of voltage-independent Ca2+ channels.     

Cytosolic Ca2+ gradients triggering unidirectional fluid secretion from exocrine pancreas.

Exocrine gland cells secrete Cl(-)-rich fluid when stimulated by neurotransmitters or hormones. This is generally ascribed to a rise in cytosolic Ca2+ concentration ([Ca2+]i), which leads to activation of Ca2(+)-dependent ion channels. A precise understanding of Cl- secretion from these cells has been hampered by a lack of knowledge about the spatial distribution of the Ca2+ signal and of the Ca2(+)-dependent ion channels in the secreting epithelial cells. We have now used the whole-cell patch-clamp method and digital imaging of [Ca2+]i to examine the response of rat pancreatic acinar cells to acetylcholine. We found a polarization of [Ca2+]i elevation and ion channel activation, and suggest that this comprises a novel 'push-pull' mechanism for unidirectional Cl- secretion. This mechanism would represent a role for cytosolic Ca2+ gradients in cellular function. The cytosolic [Ca2+]i gradients and oscillations of many other cells could have similar roles.     

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.     

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.     

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.