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.     

Topographical rearrangement of acetylcholine receptors alters channel kinetics

Plasma membranes are dynamic structures of proteins and lipids. Protein-protein or protein-lipid interactions within the membrane are believed to have important roles in many membrane functions, including ion transport, enzyme activity and signal reception. The acetylcholine (ACh) receptor-channel complex in skeletal muscle membrane is one of the best known integral membrane proteins. Its ion transport function is accessible to direct measurement at the single-channel level by the use of the 'giga-seal' patch recording technique. Here we used an in situ electrophoresis technique to rearrange the topography of pre-existing ACh receptor-channels in the muscle membrane, and measured the single-channel kinetics of ACh-activated channels in two different molecular environments within the membrane: those in the diffusely distributed region and those in the ACh receptor clusters induced by the applied field. We found that the channel kinetics are significantly prolonged in the ACh receptor cluster compared with the non-clustered region of the same cell. This result strongly supports the notion that the function of a membrane ionic channel depends on the local molecular environment.     

Single acetylcholine-activated channels show burst-kinetics in presence of desensitizing concentrations of agonist

High resolution measurements of the current through individual ion channels activated by acetylcholine (AChR- channels) in frog muscle have shown that these currents are discrete pulse-like events with durations of a few milliseconds. Fluctuation and relaxation measurements of end-plate currents have led to the conclusion that the rate of channel opening increases with agonist concentrations, and that the channel, once open, closes spontaneously. Katz and Thesleff have shown, however, that in the continued presence of ACh, the initial end-plate current declines to an equilibrium value with a time constant of several seconds. This reversible phenomenon is referred to as receptor desensitization. We report here that in the presence of ACh concentrations sufficient to cause desensitization, single channel current pulses appear in groups. From the temporal sequence of the pulses, we have derived estimates of the rates of activation and desensitization of the AChR-channel.     

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.     

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.     

Single Na+ channel currents observed in cultured rat muscle cells

The voltage- and time-dependent conductance of membrane Na+ channels is responsible for the propagation of action potentials in nerve and muscle cells. In voltage-step-clamp experiments on neurone preparations containing 10(4)-10(7) Na+ channels the membrane conductance shows smooth variations in time, but analysis of fluctuations and other eivdence suggest that the underlying single-channel conductance changes are stochastic, rapid transitions between 'closed' and 'open' states as seen in other channel types. We report here the first observations of currents through individual Na+ channels under physiological conditions using an improved version of the extracellular patch-clamp technique on cultured rat muscle cells. Our observations support earlier inferences about channel gating and show a single-channel conductance of approximately 18 pS.     

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.     

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 appearance of single-ion channels in unmodified lipid bilayer membranes at the phase transition temperature

Ion-specific channels in artificial membranes have been formed by the addition of gramicidin A, alamethicin, polyene antibiotics and some proteins to the solution surrounding the bilayer lipid membrane. Until now there have been no reports of single-ion channels in unmodified lipid membranes. We have now studied the electrical conductance of planar lipid bilayers membranes made of synthetic distearoylphosphorylcholine (DSPC). Current fluctuations of amplitude approximately 1pA and duration approximately 1 s have been discovered at phase transition temperature, which shows that the appearance of ionic channels may be the result of lipid domain interactions. This would explain the dramatic increase in ion permeability observed in liposomes during phase transition. We suggest that these channels could conduct the transmembrane ionic current in biological membranes without the involvement of peptides and proteins.     

cGMP-dependent cation channel of retinal rod outer segments

Light-modulated cytoplasmic cGMP simultaneously controls plasma membrane Na+ conductance in visual excitation and Ca2+ entry into rods by direct interaction with the cation channel. Cytoplasmic Ca2+ in turn may set operating points and contribute to the dynamics of several enzymes that regulate cGMP levels in the dark, recovery from excitation and receptor adaptation or down regulation. Similar channels may couple electrical activity to internal nucleotide metabolism in other tissues. We here report the identification, partial purification and behaviour after reconstitution of a protein of relative molecular mass 39,000 (Mr 39K) present in both disk and plasma membranes from bovine rod outer segments that mediates these cGMP-dependent cation fluxes. Its cGMP agonist specificity, kinetic cooperativity, ionic selectivity, membrane density and other features closely match the properties of the visual cGMP-dependent conductance inferred from electrophysiological measurements.     

Existence of distinct sodium channel messenger RNAs in rat brain

The sodium channel is a voltage-gated ionic channel essential for the generation of action potentials. It has been reported that the sodium channels purified from the electric organ of Electrophorus electricus (electric eel) and from chick cardiac muscle consist of a single polypeptide of relative molecular mass (Mr) approximately 260,000 (260K), whereas those purified from rat brain and skeletal muscle contain, in addition to the large polypeptide, two or three smaller polypeptides of Mr 37-45K. Recently, we have elucidated the primary structure of the Electrophorus sodium channel by cloning and sequencing the DNA complementary to its messenger RNA. Despite the apparent homogeneity of the purified sodium channel preparations, several types of tetrodotoxin (or saxitoxin) binding sites or sodium currents have been observed in many excitable membranes. The occurrence of distinguishable populations of sodium channels may be attributable to different states of the same channel protein or to distinct channel proteins. We have now isolated complementary DNA clones derived from two distinct rat brain mRNAs encoding sodium channel large polypeptides and present here the complete amino-acid sequences of the two polypeptides (designated sodium channels I and II), as deduced from the cDNA sequences. A partial DNA sequence complementary to a third homologous mRNA from rat brain has also been cloned.     

A Cl- conductance activated by hyperpolarization in Aplysia neurones

Although many voltage-gated cation channels have been described and extensively studied in biological membranes, there are very few examples of voltage-gated anion channels. Chloride conductances activated by depolarization have been observed in skate electroplaque and in frog and chick skeletal muscle. A Cl- conductance activated by hyperpolarization has been suggested both for frog muscle treated with acid (pH 5) solutions, and for crayfish muscle where it could account for the fact that the pronounced inward-going rectification of the I-V curve disappears if the fibres have been soaked in a Cl(-)-free solution. More recently, voltage-dependent anion channels extracted from biological membranes have been incorporated into artificial membranes. I now report that in Aplysia neurones, and in particular those in which the internal Cl- concentration has been increased, a Cl- conductance can be observed which is slowly activated by hyperpolarization and shows a vary steep voltage dependence. This time- and voltage-dependent Cl- conductance probably exists also in many other cells. Its presence might explain why it is difficult when using KCl-filled microelectrodes to maintain prolonged hyperpolarizations. This Cl- conductance constitutes a new type of inward-going rectification distinct both from the classical "anomalous rectification' which involves selective K+ channels and from the current termed if in heart muscle that is presently attributed to a cationic conductance.     

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.     

Interference between two indistinguishable electrons from independent sources

Very much like the ubiquitous quantum interference of a single particle with itself, quantum interference of two independent, but indistinguishable, particles is also possible. For a single particle, the interference is between the amplitudes of the particle's wavefunctions, whereas the interference between two particles is a direct result of quantum exchange statistics. Such interference is observed only in the joint probability of finding the particles in two separated detectors, after they were injected from two spatially separated and independent sources. Experimental realizations of two-particle interferometers have been proposed; in these proposals it was shown that such correlations are a direct signature of quantum entanglement between the spatial degrees of freedom of the two particles ('orbital entanglement'), even though they do not interact with each other. In optics, experiments using indistinguishable pairs of photons encountered difficulties in generating pairs of independent photons and synchronizing their arrival times; thus they have concentrated on detecting bunching of photons (bosons) by coincidence measurements. Similar experiments with electrons are rather scarce. Cross-correlation measurements between partitioned currents, emanating from one source, yielded similar information to that obtained from auto-correlation (shot noise) measurements. The proposal of ref. 3 is an electronic analogue to the historical Hanbury Brown and Twiss experiment with classical light. It is based on the electronic Mach-Zehnder interferometer that uses edge channels in the quantum Hall effect regime. Here we implement such an interferometer. We partitioned two independent and mutually incoherent electron beams into two trajectories, so that the combined four trajectories enclosed an Aharonov-Bohm flux. Although individual currents and their fluctuations (shot noise measured by auto-correlation) were found to be independent of the Aharonov-Bohm flux, the cross-correlation between current fluctuations at two opposite points across the device exhibited strong Aharonov-Bohm oscillations, suggesting orbital entanglement between the two electron beams.     

Calcium entry through stretch-inactivated ion channels in mdx myotubes.

Recent advances in understanding the molecular basis of human X-linked muscular dystrophies have come from the identification of dystrophin, a cytoskeletal protein associated with the surface membrane. Although there is little or virtually no dystrophin in affected individuals, it is not known how this causes muscle degeneration. One possibility is that the membrane of dystrophic muscle is weakened and becomes leaky to Ca2+. In muscle from mdx mice, an animal model of the human disease, intracellular Ca2+ is elevated and associated with a high rate of protein degradation. The possibility that a lack of dystrophin alters the resting permeability of skeletal muscle to Ca2+ prompted us to compare Ca2(+)-permeable ionic channels in muscle cells from normal and mdx mice. We now show that recordings of single-channel activity from mdx myotubes are dominated by the presence of Ca2(+)-permeable mechano-transducing ion channels. Like similar channels in normal skeletal muscle, they are rarely open at rest, but open when the membrane is stretched by applying suction to the electrode. Other channels in mdx myotubes, however, are often open for extended periods of time at rest and close when suction is applied to the electrode. The results show a novel type of mechano-transducing ion channel in mdx myotubes that could provide a pathway for Ca2+ to leak into the cell.     

Augmentation of cardiac calcium current by flash photolysis of intracellular caged-Ca2+ molecules

The entry of calcium ions into cells through voltage-activated Ca2+ channels in the plasma membrane triggers many important cellular processes. The activity of these channels is regulated by several hormones and neurotransmitters, as well as intracellular messengers such as Ca2+ itself (for examples, see refs 1-9). In cardiac muscle, myoplasmic Ca2+ has been proposed to potentiate Ca2+ influx, although a direct effect of Ca2+ on these channels has not yet been demonstrated. Photosensitive 'caged-Ca2+' molecules such as nitr-5, however, provide powerful tools for investigating possible regulatory roles of Ca2+ on the functioning of Ca2+ channels. Because its affinity for Ca2+ is reduced by irradiation, nitr-5 can be loaded into cells and induced to release Ca2+ with a flash of light. By using this technique we found that the elevation of intracellular Ca2+ concentration directly augmented Ca2+-channel currents in isolated cardiac muscle cells from both frog and guinea pig. The time course of the current potentiation was similar to that seen with beta-adrenergic stimulation. Thus Ca2+ may work through a similar pathway, involving phosphorylation of a regulatory Ca2+-channel protein. This mechanism is probably important for the accumulation of Ca2+ and the amplification of the contractile response in cardiac muscle, and may have a role in other excitable cells.     

Transmitter-like action of ATP on patched membranes of cultured myoblasts and myotubes

The concept of purinergic neurotransmission, first proposed by Burnstock, has been confirmed in various cell types. We show here, by the patch-clamp method, that external ATP in micromolar concentrations (1-100 microM) activates cation channels in the membranes of fusion-competent myoblasts and myotubes. In cell-attached membrane patches of myoblasts and myotubes the mean number of simultaneously activated channels increases with time after external ATP application. In myoblasts only one population of channels having a mean single-channel conductance of gamma=43 pS was found, while in myotubes two populations with gamma 1=48 pS and gamma 2=20 pS were observed. Treatment of myotube membranes with acetylcholine (ACh) or carbachol resulted in two populations of channels which had conductance values and voltage-dependent mean channel lifetimes similar to those produced in response to ATP. The results show that embryonic skeletal muscle cells contain cation channels sensitive to ATP and provide evidence for a neurotransmitter-like action of ATP on these cells.     

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.     

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.     

Glutamate activates multiple single channel conductances in hippocampal neurons

There is considerable evidence that glutamate is the principal neurotransmitter that mediates fast excitatory synaptic transmission in the vertebrate central nervous system. This single transmitter seems to activate two or three distinct types of receptors, defined by their affinities for three selective structural analogues of glutamate, NMDA (N-methyl-D-aspartate), quisqualate and kainate. All these agonists increase membrane permeability to monovalent cations, but NMDA also activates a conductance that permits significant calcium influx and is blocked in a voltage-dependent manner by extracellular magnesium. Fast synaptic excitation seems to be mediated mainly by kainate/quisqualate receptors, although NMDA receptors are sometimes activated. We have investigated the properties of these conductances using single-channel recording in primary cultures of hippocampal neurons, because the hippocampus contains all subtypes of glutamate receptors and because long-term potentiation of synaptic transmission occurs in this structure. We find that four or more distinct single-channel currents are evoked by applying glutamate to each outside-out membrane patch. These conductances vary in their ionic permeability and in the agonist most effective in causing them to open. Clear transitions between all the conductance levels are observed. Our observations are compatible with the model that all the single channel conductances activated by glutamate reflect the operation of one or two complex molecular entities.