Sarcoplasmic reticulum contains adenine nucleotide-activated calcium channels

Rapid calcium efflux from the sarcoplasmic reticulum (SR) is a necessary step in excitation-contraction coupling in skeletal muscle and is thought to be mediated by a calcium channel. Calcium efflux has been studied in fragmented SR vesicles by radioisotope efflux and fluorescence measurements. Several laboratories have reported that adenine nucleotides can stimulate calcium efflux from SR. In recent reports, Ca2+ release with a first-order rate constant as high as 100 s-1 has been observed for nucleotide-stimulated Ca2+ release from SR vesicles. Also, radioisotope efflux was blocked by Mg2+ and micromolar concentrations of the polycationic dye, ruthenium red. These high rates of transport are difficult to reconcile with a mechanism other than passive diffusion through a nucleotide-activated 'calcium release channel'. Using the fusion technique for inserting SR proteins into planar lipid bilayers, we report here single-channel recordings of calcium release channels from purified 'heavy' SR membranes. Channels have been identified on the basis of their activation by adenine nucleotides, blockade by ruthenium red, and selectivity for divalent cations. Surprisingly, the channel studied here exhibits an unusually large conductance of 170 pS in 50 mM Ba2+ while still being capable of discriminating against monovalent cations by a permeability ratio, P(Ba)/P(Cs) = 11.4.     

Inositol 1,4,5-trisphosphate activates a channel from smooth muscle sarcoplasmic reticulum

Inositol 1,4,5-trisphosphate (InsP3) can initiate calcium release into the cytoplasm in a variety of cells. From experiments using permeabilized cells, membrane vesicles, and patch-clamp techniques, it has been suggested that InsP3 acts by directly opening calcium channels. Here, we show that InsP3 induced openings of channels in planar lipid bilayers into which vesicles made from aortic muscle sarcoplasmic reticulum (SR) were incorporated. Activation of channels by InsP3 was not observed when vesicles made from SR of cardiac or skeletal muscle were incorporated into planar lipid bilayers. The present study demonstrates for the first time unique properties of an InsP3-gated calcium channel in sarcoplasmic reticulum vesicles from vascular smooth muscle. This InsP3-activated channel from aortic SR differs strikingly from the calcium-gated calcium channel of striated muscle SR in single-channel conductance and pharmacology.     

Calcium channels in thrombin-activated human platelet membrane

Platelet-activating factor, 5-hydroxytryptamine, thromboxane A2, adenosine diphosphate and thrombin are known to activate platelets by stimulating calcium entry, but the nature of the entry pathways is unknown. We present the identification of single divalent cation channels from thrombin-activated human platelets. Membrane vesicles from unstimulated and thrombin-stimulated human platelets were incorporated in planar bilayers and unitary currents through single channels were measured. Divalent cation selective channels could only be demonstrated in thrombin-stimulated preparations. These channels share a number of properties in common with voltage-dependent calcium channels--a high degree of selectivity for divalent cations, a single channel conductance of about 10 pS (in 150 mM Ba2+) and sensitivity to blockade by inorganic calcium channel blockers such as Ni2+. In other respects, these channels are different as they are not voltage-dependent and are not blocked by 1,4-dihydropyridine calcium channel antagonists.     

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.     

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.     

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.     

Noradrenaline contracts arteries by activating voltage-dependent calcium channels

Noradrenaline (NA) regulates arterial smooth muscle tone and hence blood vessel diameter and blood flow. NA apparently increases tone by causing a calcium influx through the cell membrane. Two calcium influx pathways have been proposed: voltage-activated calcium channels and NA-activated calcium-permeable channels that are voltage-insensitive. Although voltage-activated calcium channels have been identified in arterial smooth muscle, voltage-insensitive calcium channels activated by NA have not. We show here that NA contractions of rabbit mesenteric arteries increase with depolarization. The increase parallels the elevation of open-state probability (P0) of single, voltage-dependent calcium channels. The action of noradrenaline can be explained by NA-activating voltage-dependent calcium channels, rather than by opening a second type of channel. We show directly that NA increases the open-state probability of single calcium channels. Thus, in the presence of NA, calcium entry through voltage-dependent calcium channels can regulate smooth muscle tone at physiological membrane potentials. These results may have relevance to pathophysiological conditions such as hypertension.     

Purification and reconstitution of the calcium release channel from skeletal muscle

The calcium release channel from rabbit muscle sarcoplasmic reticulum (SR) has been purified and reconstituted as a functional unit in lipid bilayers. Electron microscopy reveals the four-leaf clover structure previously described for the 'feet' that span the transverse tubule (T)-SR junction. Ca2+ release from the SR induced by T-system depolarization during excitation-contraction coupling in muscle may thus be effected through a direct association of the T-system with SR Ca2+-release 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.     

Alteration of ionic selectivity of a K+ channel by mutation of the H5 region

The high ionic selectivity of K+ channels is a unifying feature of this diverse class of membrane proteins. Though K+ channels differ widely in regulation and kinetics, physiological studies have suggested a common structure: a single file pore containing multiple ion-binding sites and having broader vestibules at both ends. We have used site-directed mutagenesis and single-channel recordings to identify a molecular region that influences ionic selectivity in a cloned A-type K+ channel from Drosophila. Single amino-acid substitutions in H5, the fifth hydrophobic region, enhanced the passage of NH4+ and Rb+, ions with diameters larger than K+, without compromising the ability of the channel to exclude the smaller cation, Na+. The mutations that substantially altered selectivity had little effect on the gating properties of the channel. We conclude that the H5 region is likely to line the pore of the K+ channel.     

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.     

A large anion-selective channel has seven conductance levels

Ion channels have generally been found to have two predominant conductance levels thought to be associated with 'open' and 'closed' states, but intermediate (subconductance) states have also been reported. We have now found that a large conductance, anion-selective channel in pulmonary alveolar epithelial cells can adopt any of six open levels of conductance that are integer multiples of 60-70 pS. The channel is usually either fully open or fully closed. The frequencies of the different conductance levels are inconsistent with the notion that there are six independent channels. We suggest that the channel consists of six conducting pathways in parallel, 'co-channels', with a shared gating mechanism that can synchronously render all of them non-conducting. Other channels with lower maximum conductance may operate in a similar way but multiple conductance levels would not easily be detected because of a less favourable signal-to-noise ratio.     

Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum.

Release of calcium from intracellular stores occurs by two pathways, an inositol 1,4,5-trisphosphate (InsP3)-gated channel and a calcium-gated channel (ryanodine receptor). Using specific antibodies, both receptors were found in Purkinje cells of cerebellum. We have now compared the functional properties of the channels corresponding to the two receptors by incorporating endoplasmic reticulum vesicles from canine cerebellum into planar bilayers. InsP3-gated channels were observed most frequently. Another channel type was activated by adenine nucleotides or caffeine, inhibited by ruthenium red, and modified by ryanodine, characteristics of the ryanodine receptor/channel6. The open probability of both channel types displayed a bell-shaped curve for dependence on calcium. For the InsP3-gated channel, the maximum probability of opening occurred at 0.2 microM free calcium, with sharp decreases on either side of the maximum. Maximum activity for the ryanodine receptor/channel was maintained between 1 and 100 microM calcium. Thus, within the physiological range of cytoplasmic calcium, the InsP3-gated channel itself allows positive feedback and then negative feedback for calcium release, whereas the ryanodine receptor/channel behaves solely as a calcium-activated channel. The existence in the same cell of two channels with different responses to calcium and different ligand sensitivities provides a basis for complex patterns of intracellular calcium regulation.     

Opening of dihydropyridine calcium channels in skeletal muscle membranes by inositol trisphosphate

In many non-muscle cells, D-inositol 1,4,5-trisphosphate (InsP3) has been shown to release Ca2+ from intracellular stores, presumably from the endoplasmic reticulum. It is thought to be a ubiquitous second messenger that is produced in, and released from, the plasma membrane in response to extracellular receptor stimulation. By analogy, InsP3 in muscle cells has been postulated to open calcium channels in the sarcoplasmic reticulum (SR) membrane, which is the intracellular Ca2+ store that releases Ca2+ during muscle contraction. We report here that InsP3 may have a second site of action. We show that InsP3 opens dihydropyridine-sensitive Ca2+ channels in a vesicular preparation of rabbit skeletal muscle transverse tubules. InsP3-activated channels and channels activated by a dihydropyridine agonist in the same preparation have similar slope conductance and extrapolated reversal potential and are blocked by a dihydropyridine antagonist. This suggests that in skeletal muscle, InsP3 can modulate Ca2+ channels of transverse tubules from plasma membrane, in contrast to the previous suggestion that the functional locus of InsP3 is exclusively in the sarcoplasmic reticulum membrane.     

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.     

Single-channel currents in isolated patches of plasma membrane from basal surface of pancreatic acini

Precise localization and characterization of conductance pathways in glandular epithelia have so far proved difficult. The patch-clamp technique for high resolution current recording, which has already been applied successfully to a number of electrically excitable cells, can in principle overcome these difficulties. We now report measurements of single-channel currents from isolated patches of plasma membrane (inside-out) from the baso-lateral surface of collagenase-isolated rat and mouse pancreatic acini. We have identified a cation channel having a conductance of approximately 30 pS and a mean open time in the range 0.3-1 s which is dependent on internal calcium. The single-channel current-voltage relationship is linear and the mean open time independent of the membrane potential. These channels may, at least in part, account for the Ca2+-mediated neural and hormonal control of pancreatic acinar membrane conductance, which is probably responsible for the Ca2+-dependent acinar fluid secretion.     

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.     

Conductances of single ion channels opened by nicotinic agonists are indistinguishable

Hypotheses concerning the mechanism by which acetylcholine-like agonists cause ion channels to open often suppose that the receptor-ionophore complex can exist in either of two discrete conformations, open and shut. On the basis of noise analysis it has been reported that certain agonists open ion channels of lower conductance than usual, though many potent agonists give similar conductances, and hence that differences in the conductance of ion channels opened by different agonists may contribute to differences in efficacy. Here we have reinvestigated this question by recording single ion channel currents evoked by acetylcholine-like agonists on embryonic rat muscle in tissue culture and on adult frog muscle endplate. Ten different agonists (Fig. 1) were tested, including several that noise analysis has suggested have a low conductance. The single-channel conductance was found to be the same, within a few per cent, for all 10 agonists. It seems that noise analysis has given erroneously low conductances in some cases. Therefore efficacy differences do not depend on differences in single-channel conductance evoked by various agonists but presumably on the position of the open-shunt equilibrium of the agonist-channel complexes.     

Defective regulation of outwardly rectifying Cl- channels by protein kinase A corrected by insertion of CFTR.

Cystic fibrosis (CF) is a lethal genetic disease resulting in a reduced Cl- permeability, increased mucous sulphation, increased Na+ absorption and defective acidification of lysosomal vesicles. The CF gene encodes a protein (the cystic fibrosis transmembrane conductance regulator, CFTR) that can function as a low-conductance Cl- channel with a linear current-voltage relationship whose regulation is defective in CF patients. Larger conductance, outwardly rectifying Cl- channels are also defective in CF and fail to activate when exposed either to cyclic AMP-dependent protein kinase A or to protein kinase C. The role of the outwardly rectifying Cl- channel in CF has been questioned. We report here that expression of recombinant CF genes using adeno-associated virus vectors in CF bronchial epithelial cells corrects defective Cl- secretion, that it induces the appearance of small, linear conductance Cl- channels, and restores protein kinase A activation of outwardly rectifying Cl- channels. These results re-establish an involvement of outwardly rectifying Cl- channels in CF and suggest that CFTR regulates more than one conductance pathway in airway tissues.     

Role for mouse macrophage IgG Fc receptor as ligand-dependent ion channel

The interaction of ligands with the mouse macrophage Fc receptor which binds IgG2b and IgG1 immune complexes (FcR gamma 2b/gamma 1) triggers phagocytosis and secretion of various mediators of inflammation. FcR gamma 2b/gamma 1 has been purified using a monoclonal anti-FcR antibody, 2.4G2, and seems to be an integral membrane glycoprotein of molecular weight (Mr) 47,000-60,000 (ref. 6). Monoclonal antibody 2.4G2 is suitable as a tool for functional studies of FcR because it binds to a functional site of the receptor and induces cellular responses that are normally associated with the occupied receptor. We reported previously that binding of ligands to the macrophage FcR resulted in Na+/K+ ion fluxes through the plasma membrane, and that similar ion fluxes were observed in proteoliposomes containing reconstituted FcR. We have now incorporated FcR into planar lipid bilayers and report here that FcR gamma 2b/gamma 1 forms ligand-dependent cation-selective ion channels, with a conductance of 60 pS in 1 M KCl and an average open channel lifetime of 250 ms. The conductance decays to baseline levels within a few minutes. These results suggest a receptor-ionophore model for the signalling of phagocytosis and inflammatory responses.