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L J DeFelice

Publications and source records attributed to L J DeFelice.

At least 37 records · Page 2Linked to original sources
PubMed1995

Sodium-dependent norepinephrine-induced currents in norepinephrine-transporter-transfected HEK-293 cells blocked by cocaine and antidepressants.

Transport of norepinephrine (NE+) by cocaine- and antidepressant-sensitive transporters in presynaptic terminals is predicted to involve the cotransport of Na+ and Cl-, resulting in a net movement of charge per transport cycle. To explore the relationship between catecholamine transport and ion permeation through the NE transporter, we established a human norepinephrine transporter (hNET) cell line suitable for biochemical analysis and patch-clamp recording. Stable transfection of hNET cDNA into HEK-293 (human embryonic kidney) cells results in lines exhibiting (1) a high number of transporter copies per cell (10(6)), as detected by radioligand binding and hNET-specific antibodies, (2) high-affinity, Na(+)-dependent transport of NE, and (3) inhibitor sensitivities similar to those of native membranes. Whole-cell voltage-clamp of hNET-293 cells reveals NE-induced, Na(+)-dependent currents blocked by antidepressants and cocaine that are absent in parental cells. In addition to NE-dependent currents, transfected cells posses an NE-independent mode of charge movement mediated by hNET. hNET antagonists without effect in non-transfected cells abolish both NE-dependent and NE-independent modes of charge movement in transfected cells. The magnitude of NE-dependent currents in these cells exceeds the expectations of simple carrier models using previous estimates of transport rates. To explain our observations, we propose that hNETs function as ion-gated ligand channels with an indefinite stoichiometry relating ion flux to NE transport. In this view, external Na+ and NE bind to the transporter with finite affinities in a cooperative fashion. However, coupled transport may not predict the magnitude or the kinetics of the total current through the transporter. We propose instead that Na+ gates NE transport and also the parallel inward flux of an indeterminate number of ions through a channel-like pore.

Adrenergic alpha-Agonists↗
PubMed1994

Inactivation of L-type Ca channels in embryonic chick ventricle cells: dependence on the cytoskeletal agents colchicine and taxol.

This article shows that colchicine and taxol strongly influence the kinetics of L-type Ca channels in intact cardiac cells, and it suggests a mechanism for this action. It is known that colchicine disassociates microtubules into tubulin, and that taxol stabilizes microtubules. We have found that colchicine increases the probability that Ca channels are in the closed state and that taxol increases the probability they are in the open state. Moreover, taxol lengthens the mean open time of Ca channels. In this regard, taxol is similar to Bay-K 8644; however, Bay K works on inside-out patches, but taxol does not. Neither colchicine nor taxol alters the number of Ca channels in a patch. We have quantified these results as follows. It is known that L-type channels in embryonic chick heart ventricle cells have voltage- and current-dependent inactivation. In 10 mM Ba, channel conductance is linear in the range -10 to 20 mV. The conductance is 12 +/- 1 pS, and the extrapolated reversal potential is 42 +/- 2 mV (n = 3). In cell-attached patches, inactivation depends on the number of channels. One channel (holding at -80 mV and stepping to 0 mV for 500 ms) shows virtually no inactivation. However, three channels inactivate with a time constant of 360 +/- 20 ms (n = 6). In similar patches, colchicine (80 microM for 15 min) decreases the inactivation time constant to 162 +/- 33 ms (n = 4) and taxol (50 microM for 10 min) virtually abolishes inactivation (time constant 812 +/- 265 ms (n = 4)). We suggest that colchicine and taxol affect Ca channels through their action on the cytoskeleton, which in turn regulates the effective concentration of inactivating ions near the mouths of channels. An alternate explanation is that free tubulin interacts directly with Ca channels.

Animals↗
PubMed1993

Calcium and potassium currents in porcine granulosa cells maintained in follicular or monolayer tissue culture.

We studied membrane currents in granulosa cells (GC), immediately after collection or after variable culture time in the everted-follicle wall or in the monolayer. GC in both systems express an inward calcium current (ICa) with T-type kinetics and voltage dependence. GC in the everted-follicle culture express an outward potassium current (IK) kinetics, which remains unchanged during three days in culture. IK has delayed-rectifier kinetics, but is insensitive to TEA, 4-AP and apamine. GC in monolayer culture develop a new, inactivating delayed-rectifier potassium current (InK), which progressively dominates as cells advance from day one to day three in culture. A similar InK was recorded in large luteal cells. A possible link between luteinization and the appearance of InK is hypothesized.

Animals↗
PubMed1993

A comparison of K+ channel characteristics in human T cells: perforated-patch versus whole-cell recording techniques.

Standard whole-cell records using the patch-clamp technique are obtained after rupturing the cell membrane just below the patch pipette. Inherent problems, such as the disruption of cellular architecture and the displacement of cytosol, are unavoidable. In the present report, a whole-cell recording technique which makes use of a monovalent cation ionophore, nystatin, was applied to lymphocytes. Nystatin-perforated patches allow electrical access to the cell interior while virtually blocking the diffusion of cellular constituents into the electrode. By comparing standard whole-cell and perforated-patch techniques we observed marked differences in: activation, inactivation, and deactivation kinetics; steady-state inactivation; and the conductance-voltage relationship of K+ currents in activated human T cells.

Adult↗
PubMed1993

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

The ability of Ca ions to inhibit Ca channels presents one of the most intriguing problems in membrane biophysics. Because of this negative feedback, Ca channels can regulate the current that flows through them. The kinetics of the channels depend on voltage, and, because the voltage controls the current, a strong interaction exists between voltage dependence and Ca dependence. In addition to this interaction, the proximity of pores and the local concentration of ions also determine how effectively the Ca ions influence channel kinetics. The present article proposes a model that incorporates voltage-dependent kinetics, current-dependent kinetics, and channel clustering. We have based the model on previous voltage-clamp data and on Ca and Ba action currents measured during the action potential in beating heart cells. In general we observe that great variability exists in channel kinetics from patch to patch: Ba or Ca currents have low or high amplitudes and slow or fast kinetics during essentially the same voltage regime, either applied step-protocols or spontaneous cell action potentials. To explain this variability, we have postulated that Ca channels interact through shared ions. The model we propose expands on our previous model for Ba currents. We use the same voltage-dependent rate constants for the Ca currents that we did for the Ba currents. However, we vary the current-dependent rate constants according to the species of the conducting ion. The model reproduces the main features of our data, and we use it to predict Ca channel kinetics under physiological conditions. Preliminary reports of this work have appeared (DeFelice et al., 1991, Biophys. J. 59:551a; Risso et al., 1992, Biophys. J. 61:248a).

Action Potentials↗
PubMed1993

Electrical maturation of the murine oocyte: an increase in calcium current coincides with acquisition of meiotic competence.

We have used the whole-cell recording technique to compare three stages of primary and secondary oocytes from F1 hybrid mice (C57BL/6J x SJL/J): neonatal germinal vesicle (NGV) stage primary oocytes from 10- to 20-day-old, prepubescent mice; mature germinal vesicle (MGV) stage primary oocytes from 12-week-old, post-pubescent, superovulated mice; first polar body (FPB) stage secondary oocytes from 12-week-old, post-pubescent mice during the normal oestrus cycle or following superovulation. NGV, MGV and FPB oocytes all exhibit two voltage-dependent currents: an inward, rapidly activating/inactivating current, and an outward, slowly activating/non-inactivating current. In 1.5 mmol/l external Ca the average peak inward current is -2.9, -12.4 and -13.8 microA/cm2 in NGV, MGV and FPB oocytes, respectively. In 20 mmol/l Ca these currents increase and the reversal potential shifts to the right. The outward current decreases slightly with growth and development: at 40 mV test potentials, NGV oocytes have average outward currents of 8.9 microA/cm2, and MGV and FPB oocytes have currents of 5.0 and 5.5 microA/cm2, respectively. Thus, MGV oocytes express FPB current patterns. The reversal potentials, kinetics and pharmacology of the currents indicate that Ca channels carry the inward current and K channels carry the outward current. During growth in vivo a gradual depolarisation accompanies maturation. Resting potentials ranged from -45 to -30 mV in NGV oocytes to -35 to -17 mV in MGV oocytes to -20 mV to -3 mV in FPB oocytes. These data suggest that a selective increase occurs in the number of Ca channels during oocyte growth. This increase precedes nuclear maturation and coincides with the acquisition of meiotic competence.

Action Potentials↗
PubMed1993

Ethanol increases K+ conductance in human T-cells.

Ethanol increased the open probability of a K channel in cultured human T-cells. Single-channel currents were studied using the patch-clamp technique in cell-attached configuration. Ethanol increased the number of simultaneously active channels and subsequent current maxima at concentrations of 35 and 50 mM from control levels of 3.2 to 4.6 pA and 8.4 pA, respectively. Current responses were elicited by a 80 mV hyperpolarizing pulse following adjustment to the same resting potential. The channel was K-selective as determined by the reversal potentials under different Na-K gradients. When the pipette contained 155 mM K, single-channel currents had a slope conductance of 28 pS and a reversal potential of -4.3 mV. Baseline current levels often shifted in a negative direction during the application of ethanol, indicating a hyperpolarization of the membrane potential and an increase in channel activity.

Cells, Cultured↗
PubMed1992

Na channels that remain open throughout the cardiac action potential plateau.

In this paper we report the direct measurement of rare Na channel events that occur during the cardiac action potential, viz., channels that open at the upstroke and remain open throughout the plateau and early repolarization phase. The technique we use allows us to record channel activity and action potentials at the same time; thus, we are certain of when the Na channels open and when they finally close. The slow Na channels have the same voltage dependence, single-channel conductance, and TTX sensitivity as the fast Na channels, and they conduct Li. It therefore seems likely that the fast and the slow currents flow through the same channel. If this interpretation is correct, then the Na channel not only initiates the action potential but also helps to maintain its plateau. It is possible that the slow Na currents represent a separate collection of channels rather than a low-probability state of the fast Na channels. Regardless of which interpretation is correct, the present experiments allow us to assess the effect of the slow currents on action potential shape and on sustained Na entry.

Action Potentials↗
PubMed1991

Ion channels in murine nuclei during early development and in fully differentiated adult cells.

The nuclear envelope functions as a selective barrier between nucleus and cytoplasm. During cycles of cell division the nuclear envelope repeatedly disassembles and re-associates. Presumably, each cycle re-establishes the functional and structural integrity of the nuclear envelope. After repeated rounds of cell division, as occurs during differentiation, the selectivity and configuration of the envelope may change. We compare the ionic conductance and the nuclear pore density in four types of murine nuclei: germinal vesicles in oocytes, pronuclei in zygotes, nuclei from two-cell blastomeres, and somatic cell nuclei from the liver. A large-conductance ion channel is present in all nuclear envelopes. Liver cell nuclei have a greater number of these channels than those from earlier developmental stages, and they also have a higher density of nuclear pores. In this article we hypothesize an association between the ion channels and the nuclear pores.

Animals↗
PubMed1991

L-type Ca2+ currents in ascidian eggs.

We have studied Ca2+ currents in ascidian eggs using the whole-cell clamp technique. T and L components, as observed in somatic cells, are present and the L-type current predominates. Since the IV relationship for these inward currents overlap at -30 mV, separation of the two components using different voltage regimes is not feasible. Increasing external Ca2+ results in larger currents. The L-type current decreases in a dose-dependent fashion in the presence of Mn2+ and Nifedipine, while the T-type current is inhibited in Ni2+. When Ba2+ was used as the carrier ion, channel kinetics and conductance were completely altered. Considering the density and kinetics of L-type channels in unfertilized eggs it is probable they play an important role in regulating cytosolic Ca2+ during early developmental processes.

Animals↗
PubMed1991

Gating of L-type Ca2+ channels in embryonic chick ventricle cells: dependence on voltage, current and channel density.

1. L-type calcium channels in embryonic chick heart ventricle have voltage-dependent, time-variant kinetics when they conduct inward currents carried by 20 mM-Ba2+. Depolarizing the membrane from -20 to 20 mV increases mean open time from 1.4 to 4.2 ms. Mean open time increases monotonically with voltage. The single-channel conductance, 18 +/- 2 pS, is approximately linear over this voltage range, and the extrapolated reversal potential is 38 +/- 5 mV. 2. In cell-attached patches with five or more L-type Ca2+ channels in the patch, the currents elicited by 500 ms depolarizing steps, from a -80 mV holding potential, inactivate rapidly and have large tail currents. In the same patch, currents from a -40 mV holding potential are smaller, inactivate more slowly, and have practically no tail currents. 3. In cell-attached patches containing one of two L-type Ca2+ channels, currents from -80 or -40 mV are virtually identical, and they are similar to the currents from multichannel patches held at -40 mV. 4. The voltage-dependent, time-variant kinetics of individual L-type Ca2+ channels are unaltered if the patch is removed from the cell and forms an inside-out configuration. In these experiments the internal membrane was bathed with an artificial, intracellular-like solution containing no phosphorylating enzymes or substrates. 5. Cells bathed in 20 mM-Ba2+ solutions and held at -80 mV have currents with an early phase that inactivates in tens of milliseconds, a late phase that inactivates in hundreds of milliseconds, and a large, slow tail current. Currents from -40 mV have only the late phase and practically no tails. However, if the maximum current is less than 0.1 pA pF-1, records from either -80 or -40 mV are virtually identical, and they are similar to currents from cells with higher channel density held at -40 mV. Furthermore, if cells are stimulated before full recovery from inactivation, the reduced current is accompanied by slower inactivation. 6. Whole-cell currents in 1.5 mM-Ca2+ solutions are entirely abolished by addition of 20 microM-nifedipine, and they are enhanced 2-3 times by addition of 30 microM-cyclic AMP and 3 mM-ATP to the whole-cell recording electrode. The whole-cell currents in 20 mM-Ba2+ solutions are also completely blocked by 20 microM-nifedipine, regardless of kinetics or holding potential. Thus, by definition, the cells we are studying contain only L-type channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

High concentration of inositol 1,4,5-trisphosphate in sea urchin sperm.

We measured inositol 1,4,5-trisphosphate (InsP3) content of sea urchin gametes by using a specific protein binding assay, and found that a spermatozoon contains 4 x 10(-19) to 1 x 10(-18) moles of InsP3 before the acrosome reaction. Since the acrosome reaction has previously been shown to increase the InsP3 content of sperm severalfold, our measurement indicates that a spermatozoon contains at least 2 x 10(-18) moles of InsP3 at fertilization, corresponding to a concentration in the spermatozoon of about 1 mM. The threshold for activation of eggs by injection of InsP3 dissolved in a much larger volume of solution has been found to be about 3 x 10(-18) moles, corresponding to a concentration in the injectate of 1 microM. This suggests that sea urchin sperm may contain enough InsP3 to activate eggs. With an electroporation method, we also showed that sperm extract acts on eggs only from inside, consistent with a primary messenger role for InsP3.

Acrosome↗