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E Stefani

Publications and source records attributed to E Stefani.

At least 91 records · Page 5Linked to original sources
PubMed1993

Characterization of large-conductance, calcium-activated potassium channels from human myometrium.

OBJECTIVES: The purpose of our study was to detect and characterize potassium channels in the plasma membrane of smooth muscle cells from human myometrium. STUDY DESIGN: Plasma membrane vesicles were incorporated into lipid bilayers to record single potassium channel activity. RESULTS: We predominantly found a "maxi" calcium-activated potassium channel (261 picosiemens). This channel was calcium (micromoles per liter range) and voltage sensitive, highly selective for K+ over Na+ and Cs+, and was sensitive to external tetraethylammonium (dissociation constant approximately 220 mumol/L) and charybdotoxin (dissociation constant approximately 23 nmol/L). External apamin and 4-aminopyridine had no effect on this channel. Another type of potassium channel that was less frequently observed was also identified. It had a smaller conductance (142 picosiemens) and it seemed to be calcium independent (up to 50 nmol/L). CONCLUSION: Human myometrium possesses abundant "maxi" calcium-activated potassium channels. This channel shares common characteristics with other "maxi" calcium-activated potassium channels, including calcium and voltage gating, high conductance and selectivity, and channel pharmacologic profile.

Calcium↗
PubMed1993

Evidence for autoimmunity in amyotrophic lateral sclerosis.

Although the etiology and pathogenesis of ALS is unknown, increasing evidence supports a role for autoimmune mechanisms in motoneuron degeneration and death. An animal model, experimental autoimmune gray matter disease, can be induced by the inoculation of spinal cord gray matter. The experimental disease is characterized by weakness secondary to the loss of upper and lower motoneurons, accompanied by inflammatory foci within the spinal cord, and IgG at the neuromuscular junction and within UMN and LMN. In human ALS, IgG is present within the UMN and LMN, and T-lymphocytes and activated microglia have been identified within spinal cord gray matter and motor cortex. ALS IgG can passively transfer physiological changes of the neuromuscular junction to mice resulting in enhanced release of acetylcholine. The ALS IgG selectively interact with calcium channels and alter channel function. These data suggest a potential role for autoimmune mechanisms in the destruction and loss of motoneurons in ALS.

Amyotrophic Lateral Sclerosis↗
PubMed1993

Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K+ channels.

In voltage-dependent ion channels, a voltage sensor region is responsible for channel activation and an aqueous pore is responsible for ion conduction. These two processes have been traditionally considered to be independent. We describe here a mutation in the putative pore region (W434F) that completely abolishes ion conduction without affecting the gating charge of the channel. Gating currents in the nonconductive mutant were found to be identical in their kinetic and steady-state properties to those in conductive channels. Gating current measurements could be performed without subtracting pulses and in the presence of normal physiological solutions. Application of internal tetraethylammonium (an open channel blocker) induced Off charge immobilization for large depolarizations, suggesting that the internal tetraethylammonium-binding site becomes available upon depolarization. We concluded that for this mutant, although the conduction pathway is not functional, the channel can still undergo the closed-open conformation in response to voltage changes.

Animals↗
PubMed1993

Calcium current inactivation in denervated rat skeletal muscle fibres.

1. The inactivation of the calcium current (ICa) was studied in single extensor digitorum longus muscle fibres of the rat. Denervation was performed by surgically removing 6-8 mm of the sciatic nerve at the sciatic notch. Electrical recordings were carried out using the double Vaseline-gap technique. Normal fibres were used as controls. 2. The time course of the onset of ICa inactivation was studied with double pulse experiments. Denervation after 14 days slowed down the onset of the inactivation process. Two depolarizing pulses with variable interpulse potential were applied. The rate of recovery from ICa inactivation was analysed with long interpulse intervals (9 to 1 s). The time constants for ICa inactivation (tau i) at -90 mV potential were 4.3 and 2.2 in normal and 14 day-denervated fibres respectively. 3. The onset of ICa inactivation was studied with a double pulse protocol with variable duration of the first pulse with constant interval (120 ms) to the second pulse (300 ms). The plot of [ICa (pulse 2)/ICa(pulse 1)]--first pulse duration relationship was fitted with a single exponential equation. The inactivation time constant (tau h) values for normal and denervated fibres were 428 and 619 ms, respectively. 4. The h infinity-Vm relationship for denervated fibres was shifted toward more negative potentials and ICa did not fully inactivate with large prepulses. The h infinity-Vm relationship was fitted with a Boltzmann equation I/Imax = 1 - (A/[1 + (exp ((Vm1/2 - Vm)/kh))]) where Vm is the potential during the conditioning pulse and A is an amplitude factor. In normal fibres, Vm1/2 (mid-point) and kh (slope) values were -28.7 mV and 7.6 mV, respectively. In 14-day-denervated fibres they were -42.2 and 8.6 mV, respectively. 5. A temperature rise from 17 to 27 degrees C greatly increased the inactivation rate of ICa. This effect was similar in control and denervated fibres. The temperature coefficient quotient (Q10) values for ICa amplitude in normal and denervated fibres were 2.4 (n = 8) and 2.3 (n = 8), respectively. The Q10 values for the inactivation time constant (tau h) were 5.14 and 5.25, respectively. ICa decay during 1 s pulses was fitted to a single exponential function in normal fibres at 17 and 27 degrees C; the time constant values were tau h = 460 +/- 53 ms and tau h = 92 +/- 17 ms, respectively. The time constants of denervated fibres at both temperatures were tau h = 644 +/- 102 ms and 146 +/- 17 ms, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1993

The action of amyotrophic lateral sclerosis immunoglobulins on mammalian single skeletal muscle Ca2+ channels.

1. The planar phospholipid bilayer technique was used to study the T-tubule skeletal muscle dihydropyridine (DHP)-sensitive calcium (Ca2+) channel. To improve the signal-to-noise ratio, Ca2+ channel activity was recorded using both 800-50 and 500-50 mM NaCl gradients. 2. Ca2+ channels were characterized by their cation selectivity and pharmacological profile. The mean open time for channels identified by these techniques was increased by the DHP agonist Bay K 8644 (2 microM), while it was decreased by the DHP antagonist nifedipine (5 microM). Nifedipine also reduced Ca2+ channel amplitude levels. 3. Immunoglobulins G (IgG) from three amyotrophic lateral sclerosis (ALS) patients (n = 14 experiments), one myasthenia gravis (MG) patient (n = 3 experiments) and one healthy individual (n = 4 experiments), were tested on Ca2+ channel activity at a final concentration of 3 mg/ml. 4. Channel mean open time, mean closed time and time integral for the current were not modified by normal IgG (n = 4 experiments). Similarly, MG IgG did not reduce channel activity (n = 3 experiments). 5. ALS IgG reduced the mean open time of DHP-sensitive Ca2+ channel activity in twelve out of fourteen experiments. In addition, in five out of twelve experiments, ALS IgG stabilized the channel to a smaller amplitude level. 6. ALS IgG reduced Ca2+ channel activity in a side-selective fashion, probably corresponding to the external side of the channel. 7. These results suggest that ALS IgG action on DHP-sensitive Ca2+ channels is not mediated by second messengers, thus favouring a direct mechanism for interaction with the DHP receptor complex.

Amyotrophic Lateral Sclerosis↗
PubMed1993

Calcium transients in single mammalian skeletal muscle fibres.

1. We studied the transient changes in myoplasmic Ca2+ concentration under current- and voltage-clamp (double Vaseline-gap technique) in cut fibres of rat extensor digitorum longus muscle using mag-fura-2 (furaptra) as Ca2+ indicator, at 3.6-3.8 microns sarcomere length and 17 degrees C. Mag-fura-5 and fura-2 were also used in order to characterize some aspects of the Ca2+ transients. 2. The peak [Ca2+] in response to a single action potential was 4.6 +/- 0.4 microM (n = 5). The time to peak of the Ca2+ transient was 4.6 +/- 0.42 ms, with half-width of 8.2 +/- 1.5 ms, time constant of the rising phase 1.15 +/- 0.25 ms, time constant of the decaying phase 3.26 +/- 0.65 ms, and delay between action potential and Ca2+ transient 2.0 +/- 0.2 ms. 3. Ca2+ transients were studied under voltage-clamp conditions at different voltages and pulse durations. The rising phase showed a complex temporal course with a fast initial increase and a second component. Both components were separated by a plateau or a brief decrease of the Ca2+ concentration. The peak Ca2+ transient was 10.5 +/- 1.3 microM (n = 22). 4. After interrupting the pulse, Ca2+ concentration decayed exponentially. The time constant of decay of the Ca2+ transient increased with the pulse voltage and duration, reaching a maximum value at potentials more positive than +10 mV and pulses longer than 200 ms. An analysis of the decaying phases of the Ca2+ transients suggests that only the removal process operates after fibre repolarization. 5. The rate of Ca2+ release from the sarcoplasmic reticulum was calculated using the Melzer, Ríos & Schneider model. The value of 17.2 +/- 3.1 micronM ms-1 (n = 10) estimated in these calculations was intermediate between those obtained by other authors from cut frog muscles (10 microM ms-1) and intact frog fibres (100 microM ms-1) using antipyrylazo III (AP III) as the Ca2+ indicator.

Action Potentials↗
PubMed1993

Fab fragments from amyotrophic lateral sclerosis IgG affect calcium channels of skeletal muscle.

Amyotrophic lateral sclerosis (ALS) is a human disease involving upper and lower motoneurons. In this paper we studied the action of specific antigen-binding site (Fab) fragments of immunoglobulins from ALS patients on dihydropyridine (DHP)-sensitive Ca2+ channel function in situ. Ca2+ channels in single mammalian skeletal muscle fibers tested by the double Vaseline gap technique and single Ca2+ channels reconstituted into bilayer were tested in these experiments. Although the observed current-voltage relationship was not modified by the addition of Fab fragments (1.5 mg/ml), peak Ca2+ current (ICa) was significantly reduced. The effect of these Fab fragments on the peak ICa reached a stable value after 60 min of incubation. ALS Fab fragments also slowed the ICa rising phase and increased the rate of tail current deactivation. Studies with double pulses demonstrated that ICa inactivation time course, voltage dependence, and recovery were not modified by ALS Fab fragments. Fab fragments from normal subjects and heat-inactivated Fab fragments from ALS patients did not induce any modification on the charge movement and ICa. In single channel studies, ALS Fab fragments reduced channels amplitude. These data support the concept of an immunological interaction between the circulating antibodies from ALS patients and DHP-sensitive Ca2+ channels.

Amyotrophic Lateral Sclerosis↗
PubMed1993

Calcium-activated K+ channels as modulators of human myometrial contractile activity.

The role of Ca(2+)-activated potassium (KCa) channels in the regulation of membrane potential, intracellular free calcium ([Ca2+]i) and contraction was investigated in uterine smooth muscle and myometrial cells. In an immortalized human myometrial cell line, oxytocin increased [Ca2+]i and [3H]inositol phosphate formation. Relaxin attenuated the oxytocin-induced increase in [Ca2+]i. In cell-attached patches, membrane depolarization activated a large-conductance KCa channel (179 +/- 4 pS). Iberiotoxin (IbTX), a potent blocker of "maxi" KCa channels (A. Galvez, G. Gimenez-Gallego, J. P. Reuben, L. Roy-Contanciin, P. Feigenbaum, G. J. Kaczorowski, and M. L. Garcia. J. Biol. Chem. 265: 11083-11090, 1990) produced long closed events (approximately 6 min) in these channels. In agreement with this blockage, IbTX depolarized the cells by 9.8 +/- 2.8 mV and caused a dose-dependent increase in [Ca2+]i with a half-maximal effective concentration of 0.79 nM. IbTX also caused phasic contractions in human myometrial strips and increased both the frequency and force of spontaneous contractions in estrogen-primed rat myometrial strips. Moreover, myometrial contractility was also affected by 1 mM tetraethylammonium, a concentration that blocks uterine smooth muscle KCa channels when applied to the extracellular side (G. J. Perez, L. Toro, S. D. Erulkar, and E. Stefani. Am. J. Obstet. Gynecol. 168: 652-660, 1993). These results strongly suggest that the large conductance KCa channels may actively participate in the control of human myometrial cell membrane potential and [Ca2+].

Calcium↗
PubMed1992

Serum antibodies to L-type calcium channels in patients with amyotrophic lateral sclerosis.

BACKGROUND AND METHODS: Sporadic amyotrophic lateral sclerosis is a chronic, progressive degenerative disease of the motor neurons of the spinal cord and motor cortex. The cause is unknown. Recent electrophysiologic studies in animals indicate that immunoglobulins from patients with this disease alter presynaptic voltage-dependent calcium currents and calcium-dependent release of neurotransmitters. To determine whether similar interactions might be identified biochemically, we used an enzyme-linked immunosorbent assay (ELISA) to detect the reaction of serum IgG with purified complexes of L-type voltage-gated calcium channels from rabbit skeletal muscle. The results from patients with amyotrophic lateral sclerosis were compared with those obtained from patients with other types of motor neuron disease, patients with autoimmune and non-autoimmune neurologic diseases, and normal subjects. RESULTS: Serum samples from 36 of 48 patients with sporadic amyotrophic lateral sclerosis (75 percent) contained IgG that reacted with L-type calcium-channel protein, and serum reactivity on ELISA correlated with the rate of disease progression (Spearman rank-correlation coefficient, 0.62). Reactive serum was present in only 1 of 25 normal subjects and 1 of 35 control patients with no motor neuron disease. Antibodies to L-type voltage-gated calcium channels were identified in 6 of 9 patients with Lambert-Eaton syndrome, and in 3 of 15 patients with Guillain-Barré syndrome. CONCLUSIONS: Antibodies to L-type voltage-gated calcium channels are present in the serum of patients with amyotrophic lateral sclerosis, and antibody titers correlate with the rate of disease progression. Together with previous data, these results suggest a role for autoimmune mechanisms in the pathogenesis of sporadic amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis↗
PubMed1992

Internal blockade of a Ca(2+)-activated K+ channel by Shaker B inactivating "ball" peptide.

Shaker B inactivating peptide ("ball peptide", BP) interacts with Ca(2+)-activated K+ (KCa) channels from the cytoplasmic side only, producing inhibition of channel activity. This effect was reversible and dose and voltage dependent (stronger at depolarized potentials). The inhibition of KCa channels by BP cannot be mimicked by an inactive point mutation of the BP, L7E. BP binds to KCa channels in a bimolecular reaction (dissociation constant of 95 microM at +40 mV). The binding site is probably located in the internal "mouth" or conduction pathway, since both external K+ and internal tetraethylammonium relieve BP-induced inhibition. These results suggest that KCa channels possess a binding site for the BP with some properties similar to the ball receptor found in Shaker B K+ channels.

Amino Acid Sequence↗
PubMed1992

Gating currents in Shaker K+ channels. Implications for activation and inactivation models.

We have studied ionic and gating currents in mutant and wild-type Shaker K+ channels to investigate the mechanisms of channel activation and the relationship between the voltage sensor of the channel and its inactivation particle. The turn on of the gating current shows a rising phase, indicating that the hypothetical identical activation subunits are not independent. Hyperpolarizing prepulses indicate that most of the voltage-dependence occurs in the transitions between closed states. The open-to-closed transition is voltage independent, as suggested by the presence of a rising phase in the off gating currents. In Shaker channels showing fast inactivation, the off gating charge is partially immobilized as a result of depolarizing pulses that elicit inactivation. In mutant channels lacking inactivation, the charge is recovered quickly at the end of the pulse. Internal TEA mimics the inactivation particle in its behavior but the charge immobilization is established faster and is complete. We conclude that the activation mechanism cannot be due to the movement of identical independent gating subunits, each undergoing first order transitions, and that the inactivation particle is responsible for charge immobilization in this channel.

Animals↗
PubMed1992

Novel voltage clamp to record small, fast currents from ion channels expressed in Xenopus oocytes.

The present report describes a novel technique for voltage-clamping amphibian oocytes in which part of the membrane is isolated by a vaseline gap and the cytoplasmic fluid is exchanged by cutting or permeabilizing the remaining membrane. The main features of this open-oocyte, vaseline-gap voltage clamp are: (a) low current noise (1 nA at 3 kHz), (b) control of the ionic composition of both the internal and external media, (c) fast time resolution (20-100 microseconds time constant of decay of the capacity transient) and (d) stable recordings for several hours. These features allow reliable measurements of tail or gating currents and the new method is especially suitable when either of these currents must be measured to test the effects of mutations introduced into the cDNAs of cloned ion channels.

Animals↗
PubMed1992

Ca2+ current and charge movement in adult single human skeletal muscle fibres.

1. The Vaseline-gap technique was used to record calcium currents (ICa) and charge movement in single cut fibres from normal human muscle. Experiments were carried out in 2 or 10 mM-extracellular Ca2+ concentration ([Ca2+]o) and at 17 or 27 degrees C. 2. The passive electrical properties of the fibres with this technique were: membrane resistance for unit length rm = 59.4 k omega cm; longitudinal resistance per unit length ri = 4.9 M omega/cm; longitudinal resistance per unit length under the Vaseline seals re = 438 M omega/cm; specific membrane resistance Rm = 1.176 k omega cm2; input capacitance = 5.53 nF; specific membrane capacitance = 8.9 microF/cm2. 3. The maximum amplitude of ICa at 17 degrees C was: in 2 mM [Ca2+]o, -0.42 microA/microF and in 10 mM [Ca2+]o, -1.44 microA/microF. At 27 degrees C and in 10 mM [Ca2+]o, it increased to -3.04 microA/microF. The calculated temperature coefficient (Q10) for the increase in amplitude from 17 to 27 degrees C was 2.1. 4. Ca2+ permeability (PCa) was calculated using the Goldman-Katz relation; in 2 mM [Ca2+]o at 17 degrees C, PCa = 1.26 x 10(-6) cm/s; in 10 mM [Ca2+]o at 17 degrees C, PCa = 2.23 x 10(-6) cm/s; in 10 mM [Ca2+]o at 27 degrees C, PCa = 4.03 x 10(-6) cm/s. 5. The activation curve calculated from the PCa was shifted by 10 mV to positive potentials when raising [Ca2+]o from 2 to 10 mM. Increasing the temperature did not change the curve. The mid-point potentials (Va 1/2) and steepness (k) of the activation curves were: at 17 degrees C, in 2 mM [Ca2+]o, Va 1/2 = -1.53 mV and k = 6.7 mV; in 10 mM [Ca2+]o, Va 1/2 = 9.96 mV and k = 6.8 mV; at 27 degrees C and 10 mM [Ca2+]o, Va 1/2 = 11.3 mV and k = 7.7 mV. The activation time constant in 10 mM [Ca2+]o reached a plateau at potentials positive to 10 mV, with a value of 93.8 ms at 17 degrees C and 17.4 ms at 27 degrees C. The calculated Q10 was 4.5. 6. The deactivation of the current was studied from tail currents at different membrane potentials in 10 mM [Ca2+]o.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium↗
PubMed1992

Charge movement and the nature of signal transduction in skeletal muscle excitation-contraction coupling.

The mechanism of transmission remains unclear. It is possible that release is reinforced by Ca(2+)-induced activation secondary to opening of release channels by another primary mechanism. Multiple results favor some function of IP3 in EC coupling; however, there are many arguments indicating that IP3 is not the primary transmitter. DHP receptors and ryanodine receptors are known to play essential roles in the triadic junction, but the biochemical make up of the junction has not been completely established, and there may be other essential proteins. The strongest argument in favor of mechanical coupling between voltage sensor and release channel is the close proximity of the channel protein to the T membrane and the fixed stoichiometry between sensors and channels.

Animals↗
PubMed1992

Progesterone increases Ca2+ currents in myometrial cells from immature and nonpregnant adult rats.

We examined the role of ovarian steroids in regulating Ca2+ channels in rat uterine smooth muscle. Ca2+ currents (ICa) in myometrial cells from nonpregnant adult rats and immature rats injected with either estrogen or progesterone or estrogen plus progesterone were measured with the whole cell patch-clamp method. ICa was more prominent in cells from diestrous rats than in cells from estrous rats. In cells from immature rats the ICa density was significantly greater in cells from progesterone-injected rats than in cells from estrogen-injected or noninjected rats. ICa in cells from rats injected simultaneously with progesterone and the progesterone antagonist RU-486 was not significantly greater than those from noninjected rats. These increases in ICa density are not the result of changes in ICa activation kinetics or voltage dependence, since both are unaffected by steroid injection. The kinetics and voltage dependence of the ICa current in cells from immature and nonpregnant adult rats are similar, suggesting that they represent a single population of Ca2+ channels.

Aging↗
PubMed1992

Ca(2+)-activated K+ channels in pregnant rat myometrium: modulation by a beta-adrenergic agent.

The properties of Ca(2+)-activated K+ currents and channels were characterized in pregnant rat myometrium in whole cell and cell-attached patches and in lipid bilayers. Membrane depolarization of cultured myometrial cells from a holding potential of -50 to +70 mV in 10-mV steps under voltage-clamp conditions (whole cell mode) activated K+ outward currents (IK). At +70 mV, in the presence of 0.2 mM external Ca2+, the amplitude and activation time constant of IK were 15.0 +/- 2.1 microA/microF and 1.5 +/- 0.2 ms, respectively. Addition of 1 microM A23187 to the external solution increased the current from a control value of 16.0 +/- 2.0 to 67.9 +/- 9.1 microA/microF. Charybdotoxin, a blocker of Ca(2+)-activated K (KCa) channels, and a low concentration of tetraethylammonium chloride (TEA; 1 mM) decreased the amplitude of IK by 47 and 62%, respectively. In cell-attached patches from these cells, 1 microM A23187 increased the open time probability of a 143 +/- 6.0 pS K+ channel. Incorporation of plasma membrane vesicles from pregnant myometrium into lipid bilayers resulted in one predominant type of K+ channel. The unitary conductance of the K+ channel was 326 +/- 9.0 pS in symmetrical 450 mM KCl. The channel activation was both voltage and Ca2+ dependent. TEA inhibited the channel activity with a dissociation constant (Kd) of 378 +/- 10 microM at -60 mV or 1,477 +/- 80 microM at +60 mV. The whole cell currents were found to be stimulated by isoproterenol, a beta-adrenergic agent.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗