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L J DeFelice

Publications and source records attributed to L J DeFelice.

At least 55 records · Page 3Linked to original sources
PubMed1990

Ion channels in the nuclear envelope.

Cell nuclei are capable of partitioning a wide variety of molecules from the cytosol, including macromolecules such as proteins and RNA, and smaller peptides, amino acids, sugars and Na+ and K+ ions, all of which can be accumulated in or excluded from the nuclear domain. There are two mechanisms behind this compartmentalization: selective retention of freely diffusible molecules, and selective entry through the nuclear envelope. It is generally accepted that the nuclear envelope restricts only the larger molecules. Here we apply the patch-clamp technique to isolated murine pronuclei and show that the nuclear envelope contains K(+)-selective channels which have multiple conductance states, the maximal conductance being 200 pS. These channels, which contribute to the nuclear membrane potential, may be important in balancing the charge carried by the movement of macromolecules in and out of the nucleus.

Animals↗
PubMed1990

Ca modulates outward current through IK1 channels.

Inward-rectifier channels in cardiac cells (IK1) stabilize the resting membrane potential near the K equilibrium potential. Here we investigate the role of IK1 in the regulation of action potentials and link this to the influx of Ca during beating. Inward Ca current alters the open-channel probability of outward IK1 current. Thus Ca ions depolarize cells not only by carrying an inward current but also by blocking an outward current.

Action Potentials↗
PubMed1990

Ca channel gating during cardiac action potentials.

How do Ca channels conduct Ca ions during the cardiac action potential? We attempt to answer this question by applying a two-microelectrode technique, previously used for Na and K currents, in which we record the patch current and the action potential at the same time (Mazzanti, M., and L. J. DeFelice. 1987. Biophys. J. 12:95-100, and 1988. Biophys. J. 54:1139-1148; Wellis, D., L. J. DeFelice, and M. Mazzanti. 1990. Biophys. J. 57:41-48). In this paper, we also compare the action currents obtained by the technique with the step-protocol currents obtained during standard voltage-clamp experiments. Individual Ca channels were measured in 10 mM Ca/1 Ba and 10 mM Ba. To describe part of our results, we use the nomenclature introduced by Hess, P., J. B. Lansman, and R. W. Tsien (1984. Nature (Lond.). 311:538-544). With Ba as the charge carrier, Ca channel kinetics convert rapidly from long to short open times as the patch voltage changes from 20 to -20 mV. This voltage-dependent conversion occurs during action potentials and in step-protocol experiments. With Ca as the charge carrier, the currents are brief at all voltages, and it is difficult to define either the number of channels in the patch or the conductance of the individual channels. Occasionally, however, Ca-conducting channels spontaneously convert to long-open-time kinetics (in Hess et al., 1984, notation, mode 2). When this happens, which is about once in every 100beats, there usually appears to be only one channel in the patch. In this rare configuration, the channel is open long enough to measure its conductance in 10 Ca/ 1 Ba. The value is 8-10 pS, which is about half the conductance in Ba. Because the long openings occur so infrequently with Ca as the charge carrier, they contribute negligibly to the average Ca current at any particular time during an action potential. However, the total number of Ca ions entering during these long openings may be significant when compared to the number entering by the more usual kinetics.

Action Potentials↗
PubMed1990

Outward sodium current in beating heart cells.

This article is a study of the fast Na current during action potentials. We have investigated the outward Na current (Mazzanti, M., and L.J. DeFelice. 1987. Biophys. J. 52:95-100) in more detail, and we have asked whether it goes through the same channels associated with the rapid depolarization phase of action potentials. We address the question by patch clamping single, spontaneously beating, embryonic chick ventricle cells, using two electrodes to record the action potential and the patch current simultaneously. The chief limitation is the capacitive current, and in this article we describe a new method to subtract it. Varying the potential and the Na concentration in the patch pipette, and fitting the corrected currents to a standard model (Ebihara, L., and E.A. Johnson. 1980. Biophys. J. 32:779-790), provides evidence that the outward current is carried by the same channels that conduct the inward current. We compare the currents in beating cells to currents in nonbeating cells using whole-cell and cell-attached patch clamp recordings. The latter tend to show more positive Na reversal potentials, with the implication that internal Na is higher in beating cells. We propose that the plateau of the action potential, which is partly due to an inward Ca current, exceeds Na action current reversal potentials, and that this driving force gives rise to an outward movement of Na ions. The existence of such a current would imply that the fast repolarization phase after the upstroke of cardiac action potentials is partly due to the Na action current.

Action Potentials↗
PubMed1990

Potassium channels and the repolarization of cardiac cells.

What is the contribution of a particular potassium current to the repolarization of cardiac myocytes? The traditional answer to this question requires clamping the cells with step voltages, finding models that describe how individual currents depend on voltage and time, driving these models with action potentials to calculate the action currents, and evaluating the contribution of each pathway to repolarization from the action currents. Another method is to measure the action currents directly from beating cells. We isolated potassium channels in cell-attached patches and averaged the current over many beats. The average channel current, mean value of i(t), is a miniature version of the action current through corresponding channels in the membrane outside the patch. The time integral of this current, scaled by channel density, N, and membrane capacity, C, is the contribution of that particular pathway to the action potential: Vi(t) = -Ni integral of to mean value of i(u) du/C Using this procedure, we have found that the delayed rectifier, IK, turns on virtually without delay following the upstroke of the action potential and gradually declines during the plateau and repolarization phases, having nearly the shape of the action potential itself. The inward rectifier, IKl, may conduct little current during the plateau and is under the control of internal Ca. The traditional method of measuring action currents from step voltage-clamp records gives qualitatively similar results. Differences may arise because factors other than voltage modulate potassium currents in beating cells.

Animals↗
PubMed1988

Surface charge near the cardiac inward-rectifier channel measured from single-channel conductance.

The conductance of a channel to permeable ions depends on the number of ions near the mouth of the pore. Surface charge controls the local concentration, and impermeable cations can modify this charge. Correlating channel conductance with the concentration of impermeable cations therefore determines the local charge near the open pore. This paper presents data from cell-attached patches on embryonic chick ventricle cells, and it uses the conductance of inward-rectifier channels in the patch (in 100 mM K, with various concentrations of Na, Ca, Ba, and Mg) to estimate the local surface potential. The results indicate the presence of ionized residues near the mouth of the channel. Using the Boltzmann equation and the Gouy-Chapman relation, the surface potential due to these residues (in 100K/33Na/0Ca/0Ba/0Mg) is -40 mV, and the charge density is -0.25 e/nm2.

Animals↗
PubMed1988

K channel kinetics during the spontaneous heart beat in embryonic chick ventricle cells.

By averaging the current that passes through cell-attached patches on beating heart cells, while measuring action potentials with a whole-cell electrode, we were able to study K channels during beating. In 7-d chick ventricle in 1.3 mM K physiological solutions at room temperature, delayed-rectifier channels have three linear conductance states: 60, 30, and 15 pS. The 60 and 15 pS conductances can exist alone, but all three states may appear in the same patch as interconverting conductance levels. The delayed-rectifier conductance states have low densities (less than 10 channels per 10-microns diam cell), and all have a reversal potential near -75 mV and the same average kinetics. Outward K current through delayed-rectifier channels follows the upstroke without appreciable delay and lasts throughout the action potential. No inward current flows through delayed-rectifier channels during beating. The early outward channel has a nonlinear conductance of 18-9 pS depending on the potential. It also turns on immediately after the upstroke of the action potential and lasts on average only 50 ms. The early outward channel has an extrapolated reversal potential near -30 mV; no inward current flows during beating. The inward-rectifier has an extrapolated conductance and reversal potential of 2-3 pS and -80 mV in 1.3 mM K. Channel kinetics are independent of external K between 10 and 120 mM, and the channel conducts current only during the late repolarization and diastolic phases of the action potential. No outward current flows through inward-rectifier channels during beating. This work parallels a previous study of Na channels using similar techniques (Mazzanti, M., and L. J. DeFelice. 1987, Biophys. J. 52:95-100).

Action Potentials↗
PubMed1987

Sperm-activated currents in ascidian oocytes.

Using patch electrodes and the whole-cell recording technique to study fertilization currents in ascidian oocytes under voltage clamp, this paper shows that between -85 and 0 mV the currents are inward with an initial peak ranging from 50 to 600 pA. Voltages more positive than 0 mV inhibit initiation of the fertilization current, but by allowing the oocyte to return briefly to its resting potential fertilization occurs and fertilization currents are outward at positive potentials. By comparison with previous single-channel work, a fertilizing spermatozoon opens about 300 large-conductance channels with zero reversal potential.

Action Potentials↗
PubMed1987

Na channel kinetics during the spontaneous heart beat in embryonic chick ventricle cells.

Using cell-attached and whole-cell recording techniques simultaneously allows the measurement of Na currents during action potentials in beating heart cells. In 7-d chick ventricle, the dynamic reversal potential for Na is 30 mV, which is 20 mV less than the reversal potential in nonbeating cells. This result implies that the spontaneous activity of heart cells raises the Na concentration at the internal face of the membrane to nearly 40 mM. Fitting the Na action currents to the Hodgkin and Huxley equations shows that patches may contain from 50 to 700 Na channels, with an average density of 23 +/- 21 per micron2. Only approximately 2% of the available Na channels are open at the peak of the Na action current. This low probability is a consequence of the channels' continual inactivation during the diastolic depolarization phase.

Action Potentials↗
PubMed1987

Regulation of the Na-conducting Ca channel during the cardiac action potential.

This paper describes the kinetics of an Na-conducting channel during spontaneous action potentials in beating heart cells and during simulated action potentials driven from the patch when the cell is not beating. Since the channel conducts Na only in Ca concentrations below 10(-6) M, and since it is insensitive to tetrodotoxin and has a conductance of 100 pS in 133 mM Na, we identify it as the Ca channel conducting Na in zero Ca. By comparing the channel in beating and nonbeating cells, but under conditions in which it experiences the same voltage in both cases, we observe that: open-channel conductance is the same in beating and nonbeating cells; the channel reversal potential is 25 mV in beating cells and 50 mV in nonbeating cells; the average current peaks later in beating cells than in nonbeating cells, and it has a different time course in the two cases that is not explained by the shift in reversal potential alone; and the average Na current through Ca channels in beating cells peaks much later during the action potential than we would expect if the channel were carrying Ca. We conclude that when the Ca channel conducts Na, its kinetics and reversal potential are strongly influenced by cytoplasmic factors that accompany beating, and that its behavior is not governed by voltage alone. We also conclude that when the Ca channel conducts Na, not only are its reversal potential and conductance altered from what they would be were the channel carrying Ca, but also the channel's kinetics depend on the permeant ion. Since only the channels in the patch are in zero Ca and are conducting Na, while the Ca channels surrounding the patch are in 1.5 mM Ca and are conducting Ca, our data support the idea that it is only the Ca passing through individual channels that influences the kinetics of those same channels.

Action Potentials↗
PubMed1986

Distribution of fertilization channels in ascidian oocyte membranes.

Fertilization currents are similar in ascidian oocytes and fragments, irrespective of the size and global origin of the fragments; this result suggests that fertilization channels are distributed uniformly over the oocyte surface. Because no correlation exists between peak current and surface area of the cell, it is probable that the fertilizing spermatozoon does not open all available precursor fertilization channels, but a fixed number limited to an area around its point of entry.

Animals↗
PubMed1986

Sodium-conducting channels in cardiac membranes in low calcium.

With no Ca in the patch electrode, two kinds of channels conduct Na in spontaneously beating embryonic chick heart cells. One channel conducts Na primarily during the upstroke of the action potential and is blocked by tetrodotoxin (TTX). The other channel conducts Na primarily during the late plateau and early repolarization phase of the action potential, but only in Ca concentrations below 10(-6) M. This second channel is TTX-insensitive and has a conductance of 50 to 90 pS, depending upon the interpretation of open-channel flickering. These two Na-conducting channels correspond to the channels that normally carry the fast Na current (INa) and the slow Ca current (Isi).

Action Potentials↗
PubMed1985

Injection of a soluble sperm fraction into sea-urchin eggs triggers the cortical reaction.

Fertilization membranes form around unfertilized sea-urchin eggs after microinjection of a soluble spermatozoa fraction isosmotic with seawater. This demonstrates that the spermatozoon contains a chemical that triggers an increase in cytosolic calcium, leading to exocytosis of cortical granules. It also demonstrates that the triggering mechanism does not require an externally-activated egg-membrane process. Further experiments show that the chemical trigger is not calcium.

Animals↗
PubMed1985

Equilibrium energy analysis of freeze-fracture planes in membranes.

We have used equilibrium energy calculations to determine the most probable freeze-fracture planes in a lipid bilayer. Using a pairwise-summation computer method, we have generated numerical values for the Van der Waals potentials (electron shell repulsion, dispersion forces and electrostatic interactions) between molecules. We have compared our theoretical predictions with the experimental conclusion that the fracture planes occur normally between lipid molecules. These calculations also provide information about the composition of intramembranous particles, the potential for local clustering of single lipid types in the fluid membrane, and the importance of lipid molecules to the function of membrane proteins such as voltage-sensitive ion channels.

Calcium↗
PubMed1984

Channel currents during spontaneous action potentials in embryonic chick heart cells. The action potential patch clamp.

Single-channel currents were recorded with the cell-attached patch-clamp technique from small clusters (2-20 cells) of spontaneously beating 7-d embryo ventricle cells. Because the preparation was rhythmically active, the trans-patch potential varied with the action potential (AP). The total current through the patch membrane was the patch action current (AC). ACs and APs could be recorded simultaneously, with two electrodes, or sequentially with one electrode. Channel activity, which varied depending on the number and type of channels in the patch, was present during normal cell firing. This method can reveal the kinetics and magnitudes of the specific currents that contributed to the AP, under conditions that reflect not only the time and voltage dependence of the channels, but also environmental factors that may influence channel behavior during the AP.

Action Potentials↗
PubMed1983

Relationship between membrane excitability and single channel open-close kinetics.

We have developed a novel technique for simulating the influence of the effects of single channel kinetics on the voltage changes associated with membrane excitability. The technique uses probability distribution functions for the durations of channel open- and closed-state lifetimes, which can be calculated for any model of the ion conductance process. To illustrate the technique, we have used the Hodgkin and Huxley model of nerve membrane ion conductances to simulate channel kinetics during predetermined voltage changes, such as a voltage jump and an action potential. We have also simulated the influence of channels on voltage changes in a free running, non-voltage-clamped patch of membrane 1 micron2 or less in area. The latter results provide a direct illustration of the relationship between fluctuations of membrane excitability and fluctuations in channel open- and closed-state lifetimes.

Action Potentials↗
PubMed1982

Small signal impedance of heart cell membranes.

The electrical impedance of seven-day ventricular embryonic chick heart cell membranes maintained in tissue culture was measured under voltage clamp using the two-microelectrode voltage-clamp technique. Small sinusoidal perturbations were added to the voltage-clamp potential and the amplitude and phase of the steady-state sinusoidal response in current was recorded as a function of mean clamp potential or perturbing frequency. The experimental results are compared with two models of excitability for heart: the MNT model (McAllister, Noble & Tsien, J. Physiol. (London) 251:1-59, (1975) and the BR model (Beeler & Reuter, J. Physiol. (London) 268:177-210, 1977). The small signal impedance of heart cell membranes, in theory and experiment, shows a resonance near 1 Hz and near the threshold potential. The effect of this resonance is to increase the effective length constant of the membrane for these conditions.

Animals↗