Biomedical subjects

S Cukierman

Publications and source records attributed to S Cukierman.

32 records · Page 2Linked to original sources

PubMed1991

Reconstitution of single calcium channels from secretory granules of rat adenohypophysis in planar lipid membranes.

Free cytosolic Ca2+ ([Ca2+]i) has been demonstrated to play a crucial role in the prolactin secretory pathway. It regulates events as apparently diverse as prolactin gene transcription and the fusion of secretory granules with the plasma membrane. It is therefore important to understand the mechanisms which regulate the level of [Ca2+]i. Because prolactin secretory granule membranes have been shown to contain large amounts of Ca2+ and there is evidence that this calcium can be released independently of the granule hormone content, we have investigated the possibility that prolactin secretory granule membranes contain Ca2+ channels. When purified prolactin secretory granules were fused with an artificial phospholipid bilayer, we found a Ca2+ channel with a linear current-voltage relationship (conductance -45 pS) in symmetrical 50 mM CaCl2 solutions. Said channel had an open probability that was weakly dependent on the transmembrane potential, and a very good selectivity for calcium over chloride ions. The channel opened and closed very rapidly, when the majority of events lasting well below 25 ms. This channel could be important for the provision of high [Ca2+]i levels necessary for granule-plasma membrane fusion and could also be involved in the modulation of Ca2+ fluxes across the plasma membrane after the exocytotic release of prolactin.

Animals↗

PubMed1991

Asymmetric electrostatic effects on the gating of rat brain sodium channels in planar lipid membranes.

The effects of ionic strength (10-1,000 mM) on the gating of batrachotoxin-activated rat brain sodium channels were studied in neutral and in negatively charged lipid bilayers. In neutral bilayers, increasing the ionic strength of the extracellular solution, shifted the voltage dependence of the open probability (gating curve) of the sodium channel to more positive membrane potentials. On the other hand, increasing the intracellular ionic strength shifted the gating curve to more negative membrane potentials. Ionic strength shifted the voltage dependence of both opening and closing rate constants of the channel in analogous ways to its effects on gating curves. The voltage sensitivities of the rate constants were not affected by ionic strength. The effects of ionic strength on the gating of sodium channels reconstituted in negatively charged bilayers were qualitatively the same as in neutral bilayers. However, important quantitative differences were noticed: in low ionic strength conditions (10-150 mM), the presence of negative charges on the membrane surface induced an extra voltage shift on the gating curve of sodium channels in relation to neutral bilayers. It is concluded that: (a) asymmetric negative surface charge densities in the extracellular (1e-/533A2) and intracellular (1e-/1,231A2) sides of the sodium channel could explain the voltage shifts caused by ionic strength on the gating curve of the channel in neutral bilayers. These surface charges create negative electric fields in both the extracellular and intracellular sides of the channel. Said electric fields interfere with gating charge movements that occur during the opening and closing of sodium channels; (b) the voltage shifts caused by ionic strength on the gating curve of sodium channels can be accounted by voltage shifts in both the opening and closing rate constants; (c) net negative surface charges on the channel's molecule do not affect the intrinsic gating properties of sodium channels but are essential in determining the relative position of the channel's gating curve; (d) provided the ionic strength is below 150 mM, the gating machinery of the sodium channel molecule is able to sense the electric field created by surface changes on the lipid membrane. I propose that during the opening and closing of sodium channels, the gating charges involved in this process are asymmetrically displaced in relation to the plane of the bilayer. Simple electrostatic calculations suggest that gating charge movements are influenced by membrane electrostatic potentials at distances of 48 and 28 A away from the plane of the membrane in the extracellular sides of the channel, respectively.

Animals↗

PubMed1990

Modulation of sodium channel gating by external divalent cations: differential effects on opening and closing rates.

The effects of external divalent cations on the steady-state open probability (Po), opening and closing rates, and conductance of batrachotoxin (BTX)-activated Na channels were studied in planar lipid bilayers. External divalent cations shifted the midpoint of the Po versus membrane potential (Vm) relation (gating curve) to more depolarized potentials. Of the group IIA divalent cations tested, Ca2+ caused the largest depolarizing shift followed by Ba2+, Sr2+ and Mg2+ in that order. In contrast, the order of decreasing efficacy for block of the single channel conductance was Ca2+, Mg2+, Sr2+ and Ba2+. Thus, because it is a relatively good shifter but a poor blocker, Ba2+ was used to study the effects of divalent cations on gating. External Ba2+ decreased the Na channel opening rate and increased the closing rate. At voltages close to the midpoint of the gating curve, the opening rate decreased by a factor of 1/2.8 while the closing rate increased by 2.0-fold. Although Ba2+ would be expected to increase the external surface potential by interacting with negatively charged residues on the channel, this preferential effect on the opening rate indicates that the effects of external Ba2+ cannot be attributed solely to an increase in the potential gradient across the voltage sensing apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Effects of membrane surface charge and calcium on the gating of rat brain sodium channels in planar bilayers.

The voltage-dependent gating of single, batrachotoxin-activated Na channels from rat brain was studied in planar lipid bilayers composed of negatively charged or neutral phospholipids. The relationship between the probability of finding the Na channel in the open state and the membrane potential (Po vs. Vm) was determined in symmetrical NaCl, both in the absence of free Ca2+ and after the addition of Ca2+ to the extracellular side of the channel, the intracellular side, or both. In the absence of Ca2+, neither the midpoint (V0.5) of the Po vs. Vm relation, nor the steepness of the gating curve, was affected by the charge on the bilayer lipid. The addition of 7.5 mM Ca2+ to the external side caused a depolarizing shift in V0.5. This depolarizing shift was approximately 17 mV in neutral bilayers and approximately 25 mV in negatively charged bilayers. The addition of the same concentration of Ca2+ to only the intracellular side caused hyperpolarizing shifts in V0.5 of approximately 7 mV (neutral bilayers) and approximately 14 mV (negatively charged bilayers). The symmetrical addition of Ca2+ caused a small depolarizing shift in Po vs. Vm. We conclude that: (a) the Na channel protein possesses negatively charged groups on both its inner and outer surfaces. Charges on both surfaces affect channel gating but those on the outer surface exert a stronger influence. (b) Negative surface charges on the membrane phospholipid are close enough to the channel's gating machinery to substantially affect its operation. Charges on the inner and outer surfaces of the membrane lipid affect gating symmetrically. (c) Effects on steady-state Na channel activation are consistent with a simple superposition of contributions to the local electrostatic potential from charges on the channel protein and the membrane lipid.

Animals↗

PubMed1987

Ryanodine does not affect the potassium channel from the sarcoplasmic reticulum of skeletal muscle.

Single K channels from skeletal muscle sarcoplasmic reticulum were incorporated into artificial membranes. Ryanodine applied to either side of the membrane did not affect the gating nor the conductance properties of those channels. These results suggest that the site of action of ryanodine is limited only to the calcium channels present in the membrane of sarcoplasmic reticulum (1).

Alkaloids↗

PubMed1986

The excitability of epinephrine-induced slow responses in rabbit atrial trabeculae.

Slow responses were induced in isolated rabbit left atrial trabeculae in a medium containing 20 mM K+ and 1.8 microM epinephrine. Under these conditions, the Na+-dependent upstroke of the cardiac action potential is absent but it is possible to obtain tetrodotoxin-resistant Ca2+-action potentials (slow responses). Conventional electrophysiological techniques were employed for stimulating the preparation and measuring transmembrane potentials. Under these experimental conditions, slow responses exhibit frequency-dependent excitability changes. The excitability of epinephrine-induced slow responses increases with increasing stimulation frequency. This increase is manifested by a decrease in the latency of the slow response and also by a decrease in the stimuli requirements. In this respect, slow responses induced by epinephrine are similar to barium-induced slow responses. These observations provide further support for the hypothesis that the Ca2+-inflow during slow response electrogenesis promotes an increase in slow response excitability. These results are discussed in relation to the propagation of the slow response through the atrioventricular node under physiological conditions.

Animals↗

PubMed1985

The K+ channel of sarcoplasmic reticulum. A new look at Cs+ block.

K+-selective ion channels from mammalian sarcoplasmic reticulum were inserted into planar phospholipid bilayers, and single-channel currents measured in solutions containing Cs+. Current through this channel can be observed in symmetrical solutions containing only Cs+ salts. At zero voltage, the Cs+ conductance is approximately 15-fold lower than the corresponding K+ conductance. The open channel rectifies strongly in symmetrical Cs+ solutions, and the Cs+ currents are independent of Cs+ concentration in the range 18-600 mM. Biionic (Cs+/K+) reversal potentials are only 10 mV, showing that Cs+ is nearly as permeant as K+, though much less conductive. Addition of Cs+ to symmetrical K+ solutions reduces current through the channel in a voltage-dependent way. The results can be explained by a free energy profile in which the channel's selectivity filter acts in two ways: to provide binding sites for the conducting ions and to serve as a major rate-determining structure. According to this picture, the main difference between high-conductance K+ and low-conductance Cs+ is that Cs+ binds to an asymmetrically positioned site approximately 20-fold more tightly than does K+.

Animals↗

PubMed1985

Effects of extracellular potassium on slow response excitability in rabbit atrial trabeculae.

Small trabeculae were excised from the rabbit left atrium and exposed to 1 mmol . litre-1 Ba2+-Tyrode solution containing variable amounts of potassium (range: 10 to 25 mmol . litre-1). One end of the trabecula was stimulated by means of a suction electrode. Intracellular microelectrodes were employed for measuring membrane potentials. In presence of Ba2+, the cell depolarises and slow responses can be elicited. Slow responses thus obtained do not propagate. Slow response overshoot, maximum rate of rise and duration are depressed by increasing extracellular potassium concentrations ([K+]o). The resting potential decreases by 1 to 3 mV on changing [K+]o from 10 to 25 mmol . litre-1. The depression in slow response overshoot caused by [K+]o is compatible with an increase in the relationship between K+ and Ca2+ permeabilities as [K+]o increases. The dependence of slow response excitability on the frequency of stimulation is also depressed by [K+]o. The inhibition of slow response electrogenesis caused by extracellular potassium is an important phenomenon to consider in trying to understand the origin of certain cardiac arrhythmias.

Animals↗

PubMed1985

Modulation of slow response excitability by calcium in rabbit atrial trabeculae.

Slow responses were induced in rabbit isolated left atrial trabeculae in a modified Tyrode solution containing 15 mM-KCl and 1 mM-BaCl2. Conventional electrophysiological techniques were employed for stimulating and recording membrane potentials. Under these conditions, the excitability of the slow response depends on the past excitatory history of the preparation. As the stimulation frequency increases (range: 0.08-1 Hz) for a conditioning period of stimulation, the excitability of the slow response increases. This can be demonstrated by a decrease in stimulus requirement necessary to maintain slow response excitation following the conditioning period of stimulation. It is shown that when extracellular Ca2+ concentration is reduced to 0.5 mM, that phenomenon of frequency-dependent slow response excitability disappears but slow responses can still be elicited. Also, the addition of D-600 to 2.7 mM-Ca2+ solutions depresses both the slow response and its frequency-dependent excitability. The absence of frequency-dependent slow response excitability is not related to the depression of the slow response upstroke caused by low-Ca2+ solutions. Increasing intracellular Ca2+ concentration by exposing the preparation to low-Na+ solutions or to ouabain did not revert the observed effects in low-Ca2+ solutions. The addition of substances (5 mM-caffeine or 0.35 microM-adrenaline) that potentiate the influx of Ca2+ in low-Ca2+ solutions was found to be effective in restoring the dependence of slow response excitability on the frequency of stimulation. The increase in extracellular Ca2+ above 4 mM depressed the excitation of the slow response. Above 5.4 mM-Ca2+, the excitation of the slow response was completely inhibited while the preparation displayed continuous oscillations in transmembrane potential. The presence of a subthreshold response (subliminal response) that precedes and triggers slow response excitation is accompanied by tension development. Since the subliminal response is responsible for the changes in slow response excitability it is proposed that the Ca2+ inflow during slow response electrogenesis modulates the excitability of the slow response. Two possible physiological implications for this finding are discussed.

Action Potentials↗

PubMed1982

Frequency-dependent excitability of "membrane" slow responses of Rabbit left atrial trabeculae in the presence of Ba2+ and high K+.

Small trabeculae of rabbit left atrium immersed in TKBa solution (Tyrode with 10 mM K+ and 1 mM Ba2+) were used to study frequency dependence of "membrane" slow response excitability at long cycle lengths (greater than 1 s). In TKBa, stimuli generate graded, low-amplitude (2-15 mV) subliminal responses of variable long duration (up to 450 ms). A full all-or-none slow response is generated when a subliminal response depolarizes the membrane to about--35 mV. Subliminal response amplitude and rate of rise augment with stimulus intensity-duration product. For a fixed stimulus, the subliminal response is larger and faster at higher frequencies. Sudden changes in stimulus frequency or time course induce changes in subliminal response tha take four to eight cycles to attain steady state. For a fixed stimulus, slow response latency shortens progressively during the first few cycles after a sudden increase in frequency or when a rested preparation is excited (latency adaptation phenomenon, LAP). Slow response threshold stimulus requirements decrease during LAP (excitability hysteresis). The degree of excitability hysteresis is dependent on stimulation frequency and is more pronounced at higher frequencies. Frequency sensitivity of subliminal response (which causes frequency sensitivity of slow response excitability) is explained in terms of a transient state of enhancement set up by each stimulus. The enhanced state decays between stimuli with a half-time of approximately 4 s, thus allowing cumulative effects to become evident at rates above 0.1 Hz.

Action Potentials↗

PubMed1982