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J B Lansman

Publications and source records attributed to J B Lansman.

At least 37 records · Page 2Linked to original sources
PubMed1990

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.

Animals↗
PubMed1990

Properties of embryonic and adult muscle acetylcholine receptors transiently expressed in COS cells.

We used transient transfection in COS cells to compare the properties of mouse muscle acetylcholine receptors (AChRs) containing alpha, beta, delta, and either gamma or epsilon subunits. gamma- and epsilon-AChRs had identical association rates for binding 125I-alpha-bungarotoxin, and identical curves for inhibition of toxin binding by d-tubocurarine, but epsilon-AChRs had a significantly longer half-time of turnover in the membrane than gamma-AChRs. A myasthenic serum specific for the embryonic form of the AChR reduced toxin binding to gamma-, but not epsilon-AChRs. The gamma-AChRs had channel characteristics of embryonic AChRs, whereas the major class of epsilon-AChR channels had the characteristics of adult AChRs. Two minor channel classes with smaller conductances were also seen with epsilon-AChR. Thus, some, but not all, of the differences between AChRs at adult endplates and those in the extrasynaptic membrane can be explained by the difference in subunit composition of gamma- and epsilon-AChRs.

Animals↗
PubMed1990

Blockade of current through single calcium channels by trivalent lanthanide cations. Effect of ionic radius on the rates of ion entry and exit.

Currents flowing through single dihydropyridine-sensitive Ca2+ channels were recorded from cell-attached patches on C2 myotubes. In the presence of dihydropyridine agonist to prolong the duration of single-channel openings, adding micromolar concentrations of lanthanum (La), cerium (Ce), neodymium (Nd), gadolinium (Gd), dysprosium (Dy), or ytterbium (Yb) to patch electrodes containing 110 mM BaCl2 caused the unitary Ba2+ currents to fluctuate between fully open and shut states. The kinetics of channel blockade followed the predictions of a simple open channel block model in which the fluctuations of the single-channel current arose from the entry and exit of blocking ions from the pore. Entry rates for all the lanthanides tested were relatively insensitive to membrane potential, however, exit rates depended strongly on membrane potential increasing approximately e-fold per 23 mV with hyperpolarization. Individual lanthanide ions differed in both the absolute rates of ion entry and exit: entry rates decreased as cationic radius decreased; exit rates also decreased with cationic radius during the first part of the lanthanide series but then showed little change during the latter part of the series. Overall, the results support the idea that smaller ions enter the channel more slowly, presumably because they dehydrate more slowly; smaller ions also bind more tightly to a site within the channel pore, but lanthanide residence time within the channel approaches a maximum for the smaller cations with radii less than or equal to that of Ca2+.

Animals↗
PubMed1990

Voltage-dependent block by zinc of single calcium channels in mouse myotubes.

1. The blocking actions of Zn2+ on currents carried by Ba2+ through single dihydropyridine-sensitive Ca2+ channels were recorded from cell-attached patches on myotubes from the mouse C2 cell line. 2. Adding 100 microM-Zn2+ to the patch electrode containing 110 mM-BaCl2 produced an increase in the open channel noise, presumably arising from unresolved blocking and unblocking of the open channel by Zn2+. Adding between 200 and 1000 microM-Zn2+ to the electrode reduced the amplitude of the unitary current in a concentration-dependent manner. 3. The single-channel current-voltage (i-V) relations showed that Zn2+ reduced the amplitude of the unitary Ba2+ currents at all potentials more negative than 0 mV. A plot of the amplitude of the unitary current in the presence of Zn2+, normalized to the amplitude in its absence, showed that block of the current depended on voltage, decreasing as the patch potential was made more negative. 4. The normalized amplitudes of the unitary currents were plotted as a function of the logarithm of [Zn2+] in the electrode. The relation for currents recorded at different potentials were fitted to an expression for binding to a single site with a KD at 0 mV of approximately 500 microM. The KD changed approximately e-fold per 83 mV with hyperpolarization. The results suggest Zn2+ binds to a site located at approximately 15% of the potential drop from the surface membrane. 5. Reducing the concentration of Ba2+ in the patch electrode enhanced the steady-state block of unitary currents by Zn2+. The inverse of the unitary current was plotted as a function of [Ba2+]o in the presence and absence of Zn2+; both were linear and intersected at the ordinate, indicating Ba2+ and Zn2+ compete for a channel site. 6. The kinetics of Zn2+ block of unitary Ba2+ currents were studied by amplitude distribution analysis. As expected for a simple reaction between blocking ion and open channel, the blocking rate depended linearly on the concentration of Zn2+, while the exit rate was independent of concentration. The second-order rate coefficient for Zn2+ entry in the presence of 110 mM-BaCl2 at 0 mV was approximately 2.0 X 10(7) M-1S-1, while the exit rate was approximately 16000 s-1. 7. Both entry and exit rates increased as the membrane potential was made more negative. The entry rate increased approximately e-fold per 66 mV, while the exit rate increased approximately e-fold per 41 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Stretch-sensitive channels in developing muscle cells from a mouse cell line.

1. Recordings of single-channel activity were made from cell-attached patches on mouse C2 muscle cells at morphologically identifiable stages of myogenesis in vitro. We have identified Ca2(+)-permeable, cation-selective channels that are gated by applying suction to the patch electrode and by changes in membrane potential and have analysed single-channel properties as well as channel expression during myogenesis. 2. Single-channel activity could be detected when the membrane was held at steady negative potentials. With monovalent cations in the electrode, the single-channel current-voltage (i-V) relations were linear. The channel is permeable to Li+, Na+, K+, Rb+ and Cs+, but is not strongly selective among the monovalent cations as judged by measurements of single-channel conductance and reversal potential. 3. With 110 mM of either CaCl2 or BaCl2 as the only inward change carrier, slope conductances were approximately 13 and 24 pS and currents reversed at approximately +22 and +17 mV, respectively. The relative permeability of Ca2+ to K+ calculated from the constant-field equation was PCa/PK = approximately 2. 4. Channel openings occurred as bursts of brief openings and closings separated by much longer closed periods. Closed-time histograms were best fitted with three exponential components, while histograms of burst duration were best fitted with two exponential components, reflecting the short and long bursts in the single-channel records. 5. Applying suction to the patch electrode while recording at steady negative membrane potentials produced channel openings to discrete current levels. Mean channel open probability depended linearly on the square of the applied pressure and was greater at positive membrane potentials. The permeability of the channel to monovalent and divalent cations was indistinguishable from the spontaneous activity recorded at steady negative potentials. 6. Channel activity recorded from cell-attached patches in the absence of applied pressure depended on membrane potential increasing approximately e-fold per 38 mV with depolarization. Analysis of the kinetics of the response to membrane potential showed that the depolarization reduced the duration of the slowest component of the closed-time distribution. 7. The lanthanide cation gadolinium (Gd) reduced the amplitude of the unitary currents in a concentration-dependent manner. The amplitudes of both inward and outward currents were reduced to the same extent suggesting block is voltage-independent. Gd produced half-maximal inhibition of the unitary current at approximately 6 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Expression of functional mouse muscle acetylcholine receptors in Chinese hamster ovary cells.

CHO cells were transfected with cDNAs for all 4 subunits of the mouse muscle acetylcholine receptor (AChR) to obtain a stable cell line (CHO-AR) expressing the AChR on its surface. Immunoprecipitation experiments established that the AChR contained alpha- and beta-subunits assembled with gamma- and/or delta-subunits. In addition, one of the toxin-binding sites of the AChR was blocked by a myasthenic serum that specifically recognizes AChR containing the gamma-subunit. AChR from the CHO-AR cells had the same sedimentation rate, association rate constant for the binding of alpha-bungarotoxin (alpha BTX), and the same metabolic half-life as the AChR in myotubes of the mouse muscle cell line C2C12. Electrophysiological assay of CHO-AR cells by single-channel recording showed the presence of ACh-responsive ion channels with the characteristics of the embryonic AChR (gamma = 40 pS, tau = 5.6 msec). In some patches a smaller conductance channel was also seen that may represent partially assembled receptor. Fluorescence microscopy of fixed, permeabilized cells stained with rhodamine-alpha-BTX demonstrated both perinuclear and diffuse surface staining. The expression of fully assembled, functional mammalian muscle AChR in nonmuscle cells will allow detailed investigation of its properties and interactions with other cellular components.

Animals↗
PubMed1987

Calcium current and calcium-activated inward current in the oocyte of the starfish Leptasterias hexactis.

1. Inward currents in the immature oocyte of the starfish Leptasterias hexactis were studied with a two-micro-electrode voltage clamp. Experiments investigated the role of Ca2+ in the Na+-dependent plateau of the action potential. 2. Voltage steps more positive than -55 mV produced inward currents in normal sea water that activated and then decayed to a non-zero level with a double-exponential time course. Returning the voltage to the resting potential produced an inward tail current that relaxed slowly to zero with a time course of seconds. 3. Replacing Na+ with choline abolished the slowly decaying component as well as the slow tail current which followed the end of the voltage pulse. This suggested that inward current in Na+-containing sea water consisted of a rapidly decaying component that flowed through Ca2+ channels and a more slowly decaying component carried by Na+. 4. Ca2+ current was isolated in Na+-free sea water. Activation followed a sigmoidal time course that could be described with m2 kinetics. Inactivation during a maintained depolarization followed first-order kinetics and was voltage dependent. 5. When Ba2+ was substituted for Ca2+ as the divalent ion charge carrier, inward currents in Na+-containing sea water decayed along a single-exponential time course. The absence of a slowly decaying Na+ current in Ba2+-containing sea water suggested that Na+ current depended on Ca2+ influx. 6. The effects of altering Ca2+ influx on the time course of Na+ current were investigated. Na+ current decayed more rapidly as the test pulse potential was made more positive, while raising [Ca2+]o slowed the decaying phase without altering its dependence on membrane potential. 7. Tail currents measured after rapidly stepping the membrane potential back to the resting level consisted of a fast component associated with the closing of Ca2+ channels and a slow component that was abolished by removing Na+. 8. The variation of the amplitude of the slow component of tail current with the duration of the voltage-clamp pulse indicated that Na+ current is associated with a time-dependent component of membrane conductance. 9. Possible mechanisms for the slowly decaying Na+ current are considered. The results are discussed in relation to the idea that the conductance change to Na+ follows the time course of Ca2+ accumulation and removal from the cytoplasm.

Action Potentials↗
PubMed1986

Mechanisms of calcium channel modulation by beta-adrenergic agents and dihydropyridine calcium agonists.

Cardiac calcium channel activity is markedly increased by beta-adrenergic agents or calcium agonists such as Bay K 8644. The molecular mechanisms underlying these important modulatory effects have been studied with patch clamp techniques by several groups. This paper presents new experiments and reviews published evidence from fluctuation analysis of whole cell calcium current and unitary recordings of single calcium channel activity. Two different factors underlie the enhancement of calcium channel activity seen with beta-stimulation or cyclic AMP: (1) increased availability of calcium channels, expressed in whole cell recordings as an increase in the number of functional channels and in single channel recordings as an increase in the proportion of non-blank sweeps. (2) changes in opening probability, due to alteration of the fast kinetics of channel opening and closing. Both factors contribute to the beta-adrenergic enhancement in frog, rat, and guinea-pig ventricular cells although their quantitative importance is somewhat variable. Unlike beta-adrenergic agents, calcium agonists such as Bay K 8644 promote a mode of channel gating that is characterized by long openings and short closings, seen only rarely in control or with beta-stimulation.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells.

Single channel and whole cell recordings were used to study ion permeation through Ca channels in isolated ventricular heart cells of guinea pigs. We evaluated the permeability to various divalent and monovalent cations in two ways, by measuring either unitary current amplitude or reversal potential (Erev). According to whole cell measurements of Erev, the relative permeability sequence is Ca2+ greater than Sr2+ greater than Ba2+ for divalent ions; Mg2+ is not measurably permeant. Monovalent ions follow the sequence Li+ greater than Na+ greater than K+ greater than Cs+, and are much less permeant than the divalents. These whole cell measurements were supported by single channel recordings, which showed clear outward currents through single Ca channels at strong depolarizations, similar values of Erev, and similar inflections in the current-voltage relation near Erev. Information from Erev measurements stands in contrast to estimates of open channel flux or single channel conductance, which give the sequence Na+ (85 pS) greater than Li+ (45 pS) greater than Ba2+ (20 pS) greater than Ca2+ (9 pS) near 0 mV with 110-150 mM charge carrier. Thus, ions with a higher permeability, judged by Erev, have lower ion transfer rates. In another comparison, whole cell Na currents through Ca channels are halved by less than 2 microM [Ca]o, but greater than 10 mM [Ca]o is required to produce half-maximal unitary Ca current. All of these observations seem consistent with a recent hypothesis for the mechanism of Ca channel permeation, which proposes that: ions pass through the pore in single file, interacting with multiple binding sites along the way; selectivity is largely determined by ion affinity to the binding sites rather than by exclusion by a selectivity filter; occupancy by only one Ca ion is sufficient to block the pore's high conductance for monovalent ions like Na+; rapid permeation by Ca ions depends upon double occupancy, which only becomes significant at millimolar [Ca]o, because of electrostatic repulsion or some other interaction between ions; and once double occupancy occurs, the ion-ion interaction helps promote a quick exit of Ca ions from the pore into the cell.

Animals↗
PubMed1986

Blockade of current through single calcium channels by Cd2+, Mg2+, and Ca2+. Voltage and concentration dependence of calcium entry into the pore.

We studied the blocking actions of external Ca2+, Mg2+, Ca2+, and other multivalent ions on single Ca channel currents in cell-attached patch recordings from guinea pig ventricular cells. External Cd or Mg ions chopped long-lasting unitary Ba currents promoted by the Ca agonist Bay K 8644 into bursts of brief openings. The bursts appear to arise from discrete blocking and unblocking transitions. A simple reaction between a blocking ion and an open channel was suggested by the kinetics of the bursts: open and closed times within a burst were exponentially distributed, the blocking rate varied linearly with the concentration of blocking ion, and the unblocking rate was more or less independent of the blocker concentration. Other kinetic features suggested that both Cd2+ and Mg2+ lodge within the pore. The unblocking rate was speeded by membrane hyperpolarization or by raising the Ba concentration, as if blocking ions were swept into the myoplasm by the applied electric field or by repulsive interaction with Ba2+. Ca ions reduced the amplitude of unitary Ba currents (50% inhibition at approximately 10 mM [Ca]o with 50 mM [Ba]o) without detectable flicker, presumably because Ca ions exit the pore very rapidly following Ba entry. However, Ca2+ entry and exit rates could be resolved when micromolar Ca blocked unitary Li+ fluxes through the Ca channel. The blocking rate was essentially voltage independent, but varied linearly with Ca concentration (rate coefficient, 4.5 X 10(8) M-1s-1); evidently, the initial Ca2+-pore interaction is outside the membrane field and much faster than the overall process of Ca ion transfer. The unblocking rate did not vary with [Ca]o, but increased steeply with membrane hyperpolarization, as if blocking Ca ions were driven into the cell. We suggest that Ca is both an effective permeator and a potent blocker because it dehydrates rapidly (unlike Mg2+) and binds to the pore with appropriate affinity (unlike Cd2+). There appears to be no sharp dichotomy between "permeators" and "blockers," only quantitative differences in how quickly ions enter and leave the pore.

Animals↗
PubMed1986

Calcium channel types in cardiac myocytes: modulation by dihydropyridines and beta-adrenergic stimulation.

We used the patch clamp technique to record unitary calcium (Ca2+) channel activity in freshly dissociated ventricular myocytes from adult guinea pigs. We found two types of Ca2+ channels with distinct permeation and gating properties and different sensitivity to pharmacological agents. One channel (T-type) requires negative membrane potentials to remove inactivation. It gives rise to a transient mean current and is not affected by dihydropyridines or isoproterenol. The other Ca2+ channel (L-type) has a larger unitary barium-conductance, activates at more positive potentials and its averaged current decays much more slowly. It shows a distinct gating pattern with different gating modes, the proportion of which is drastically altered by dihydropyridine Ca2+-channel agonists and antagonists. L-type channel activity is modulated by beta-adrenergic stimulation by a mechanism of action which differs from that of dihydropyridine Ca2+-channel agonists.

Animals↗
PubMed1983

Developmental regulation of Ca2+ and K+ currents during hormone-induced maturation of starfish oocytes.

Changes in the electrical properties of starfish oocytes during hormone-induced maturation (the reinitiation of meiosis prior to fertilization) were studied by using the voltage-clamp technique. Three voltage-dependent ionic currents dominate the current-voltage relation of the immature oocyte: an inward Ca2+ current, a fast transient K+ current similar to the "A current" of molluscan neurons, and an inwardly rectifying K+ current. During in vitro maturation stimulated by the natural maturing hormone 1-methyladenine, gradual changes in the amplitudes of all three currents were seen: the Ca2+ currents became larger, and both K+ currents became smaller. The kinetics of the currents were not significantly altered during maturation. As a result of these changes, action potentials in the mature egg had lower thresholds, faster rates of rise, and larger overshoots than those of the immature oocyte. We also found that the total membrane capacitance decreased substantially during maturation, perhaps indicating a decrease in membrane surface area triggered by the hormone. The significance of these results is discussed in terms of the preparation of the immature oocyte for fertilization and the mechanisms of modification of ion channel properties during development.

Action Potentials↗
PubMed1983

Voltage-clamp study of the conductance activated at fertilization in the starfish egg.

Ionic currents underlying the fertilization potential of the egg of the starfish Mediaster aequalis were studied using a two-micro-electrode voltage clamp. Mature eggs were fertilized in vitro under voltage-clamp conditions. The fertilization current, here termed IF, was induced by adding sperm to sea water bathing the egg. At a holding potential of -70 mV, IF was inward. It reached a peak within 2-4 min and then decayed over the next approximately 20 min with a rate which depended on the holding membrane potential. Instantaneous current-voltage relations measured at different times during IF were approximately linear and reversed at a potential of +6.0 +/- 5.8 mV (mean +/- S.D., n = 11). Membrane chord conductance was highest at the peak of inward current and the declining phase of IF was due to a decrease in conductance towards the pre-fertilization level. When the membrane potential was rapidly stepped to levels more positive than about -45 mV, the conductance underlying IF decreased in a manner which depended on both membrane potential and time. The fertilization-specific conductance showed a sigmoidal activation curve between -50 and +10 mV with a half-activation level of -25 mV. Analysis of the steady-state voltage dependence indicated that at the peak of the fertilization potential (+10 to +15 mV) only 4-5% of the total available channels would be open. Current relaxations followed first-order kinetics and the relaxation time constant depended upon the membrane potential during the voltage pulse. The relation between the time constant and voltage was bell-shaped, decreasing at potentials more negative than -40 and more positive than 0 mV. Both the steady-state conductance-voltage relation and the kinetics of the current relaxations were consistent with a simple two-state gating model in which the probability of a channel being open is determined by a single gating particle with an effective valency of -1.7 moving through the entire membrane field. The shifts in reversal potential with changes in external Na (at 10 mM-external K) were analysed using the constant field expression, which gave a relative permeability of Na to K of approximately 0.6.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1982

Neurotensin stimulates exocytotic histamine secretion from rat mast cells and elevates plasma histamine levels.

1. Neurotensin stimulated histamine release and granule extrusion when applied to isolated rat peritoneal mast cells. 2. This secretory response was prevented by the removal of calcium or energy and was not accompanied by the release of lactic dehydrogenase. 3. The secretory response produced by neurotensin was prevented by prior treatment of mast cells with cromoglycate. 4. The intravenous injection of neurotensin into anaesthetized rats produced a rapid and significant increase in the level of blood histamine that was dependent upon the dose of neurotensin. 5. Treatment of rats with compound 48/80, 24 hr before neurotensin, abolished the elevation in blood histamine caused by neurotensin. The intravenous injection of cromoglycate 1-2 min before neurotensin greatly reduced the response to neurotensin. 6. The intradermal injection of neurotensin (0.03-30 p-mole) increased capillary permeability in rats pre-treated intravenously with Evans Blue. This response was abolished by the antihistamine, diphenhydramine. Increasing the dose of neurotensin to 300 p-mole partially overcame this inhibition by diphenhydramine. 7. Our results demonstrate that neurotensin can elicit an exocytotic secretory response from isolated rat peritoneal mast cells and elevate histamine levels in blood. It is suggested that some of neurotensin's physiological effects may be due to stimulation of mast cell secretion.

Animals↗
PubMed1982

Stimulus-secretion coupling in rat mast cells: inactivation of extracellular calcium dependent secretion.

1. Stimulation by compound 48/80 of mast cells deprived of Ca failed to release histamine. Secretion of histamine was elicited from such cells by the subsequent introduction of Ca. 2. Histamine secretion declined as the interval between stimulation by compound 48/80 and the introduction of Ca increased. This decline is called inactivation. 3. The addition of the ionophore, A23187, with Ca restored maximum histamine secretion overcoming inactivation. 4. Increasing the concentration of Ca introduced after stimulation, from 2 to 8 mM, or to 20 mM reduced the amount of histamine released. This reduction was proportional to the interval between stimulation and the introduction of Ca. The addition of A23187 with the higher concentrations of Ca fully restored histamine secretion. 5. Stimulation of mast cells in Ca-free media by the secretagogues polymyxin B or bradykinin, and the subsequent introduction of Ca, resulted in a similar inactivation or decline in histamine release. 6. Mast cells inactivated by compound 48/80 stimulation in Ca-free media showed no increase in histamine release when the secretagogues polymyxin B plus Ca or bradykinin plus Ca were added. However, when A23187 plus Ca was added, a full secretory response was obtained. 7. It is suggested that the process of inactivation involves time-dependent change in the Ca permeability of the mast cell membrane. The concentration of introduced Ca is suggested to influence the regulation of this permeability.

Animals↗