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G Isenberg

Publications and source records attributed to G Isenberg.

At least 109 records · Page 6Linked to original sources
PubMed1992

Gating of maxi K+ channels studied by Ca2+ concentration jumps in excised inside-out multi-channel patches (myocytes from guinea pig urinary bladder).

Currents through maxi K+ channels were recorded in inside-out macro-patches. Using a liquid filament switch (Franke, C., H. Hatt, and J. Dudel. 1987. Neurosci, Lett. 77:199-204) the Ca2+ concentration at the tip of the patch electrode ([Ca2+]i) was changed in less than 1 ms. Elevation of [Ca2+]i from less than 10 nM to 3, 6, 20, 50, 320, or 1,000 microM activated several maxi K+ channels in the patch, whereas return to less than 10 nM deactivated them. The time course of Ca(2+)-dependent activation and deactivation was evaluated from the mean of 10-50 sweeps. The mean currents started a approximately 10-ms delay that was attributed to diffusion of Ca2+ from the tip to the K+ channel protein. The activation and deactivation time courses were fitted with the third power of exponential terms. The rate of activation increased with higher [Ca2+]i and with more positive potentials. The rate of deactivation was independent of preceding [Ca2+]i and was reduced at more positive potentials. The rate of deactivation was measured at five temperatures between 16 and 37 degrees C; fitting the results with the Arrhenius equation yielded an energy barrier of 16 kcal/mol for the Ca2+ dissociation at 0 mV. After 200 ms, the time-dependent processes were in a steady state, i.e., there was no sign of inactivation. In the steady state (200 ms), the dependence of channel openness, N.P(o), on [Ca2+]i yielded a Hill coefficient of approximately 3. The apparent dissociation constant, KD, decreased from 13 microM at -50 mV to 0.5 microM at +70 mV. The dependence of N.P(o) on voltage followed a Boltzmann distribution with a maximal P(o) of 0.8 and a slope factor of approximately 39 mV. The results were summarized by a model describing Ca2+- and voltage-dependent activation and deactivation, as well as steady-state open probability by the binding of Ca2+ to three equal and independent sites within the electrical field of the membrane at an electrical distance of 0.31 from the cytoplasmic side.

Animals↗
PubMed1992

Caffeine-induced release and reuptake of Ca2+ by Ca2+ stores in myocytes from guinea-pig urinary bladder.

1. Voltage-clamped isolated smooth muscle cells from guinea-pig urinary bladder were studied with 3.6 mM extracellular Ca2+ at 36 degrees C. The fluorescence of the Ca(2+)-sensitive dye Indo-1 was used to monitor the cytosolic calcium concentration ([Ca2+]i) and its changes ([Ca2+]i transient). Fast application of caffeine (10 mM) to the cell was used to release the intracellular Ca2+ from a 'caffeine-sensitive Ca2+ store'. 2. At the holding potential -60 mV, a short (1 s) caffeine application increased [Ca2+]i within less than 1 s from the resting 118 +/- 22 nM to 1490 +/- 332 nM. Following the caffeine wash-out, [Ca2+]i fell from this peak to a subresting level of 47 +/- 12 nM, i.e. an 'undershoot' of [Ca2+]i occurred. Subsequent caffeine-induced [Ca2+]i transients had attenuated peaks suggesting that the caffeine-sensitive Ca2+ store had lost a part of the releasable Ca2+. 3. In the continuous presence of caffeine, [Ca2+]i decayed from its peak to control resting [Ca2+]i values. The wash-out of caffeine following prolonged (10-30 s) treatment also resulted in [Ca2+]i undershoot. Subsequent caffeine-induced [Ca2+]i transients were largely abolished as if the caffeine-sensitive Ca2+ store had lost a large part of releasable Ca2+. During the undershoot, hyperpolarization to -100 mV did not affect [Ca2+]i. In most cells studied, recovery of [Ca2+]i from the undershoot to the resting level required depolarizations inducing Ca2+ influx through L-type Ca2+ channels. 4. Block of plasmalemmal Ca(2+)-ATPase (PMCa) with extracellular La3+ (3 mM) did not modify the decay of the [Ca2+]i transients induced by depolarization or by a 1 s caffeine application suggesting that decay rate of both is not limited by PMCa rate. La3+ abolished the undershoot of [Ca2+]i. In the continuous presence of caffeine, La3+ largely prevented the decay of [Ca2+]i. 5. When the depolarizing steps from -60 to 0 mV (160 ms duration) were applied during the period of [Ca2+]i undershoot, the half-time of decay of the corresponding [Ca2+]i transients was up to three times faster than in control. Repetitive depolarizations restored the rate of decay and [Ca2+]i recovered to the resting value. Both processes recovered along a similar time course. 6. Application of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 0.1 mM) or of 8-Br-cAMP (0.1 mM) did not mimic the above caffeine effects suggesting that stimulation of sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCa) by cAMP-dependent phosphorylation is not the underlying mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1992

Contribution of Ca(2+)-induced Ca2+ release to the [Ca2+]i transients in myocytes from guinea-pig urinary bladder.

1. Smooth muscle cells from guinea-pig urinary bladder were studied at an extracellular Ca2+ concentration ([Ca2+]o) of 3.6 mM and 36 degrees C. Fluorescence of Indo-1 was used to monitor the cytosolic calcium concentration ([Ca2+]i) and its changes ([Ca2+]i transients) induced by step membrane depolarizations. 2. During a 6 s depolarization step from -60 to 0 mV [Ca2+]i increased from a resting 118 +/- 22 nM to 1150 +/- 336 nM and decayed to a sustained level of 295 +/- 62 nM. The experiments were designed to evaluate the contribution of the release of intracellularly stored Ca2+ to components of the depolarization-induced [Ca2+]i transient, i.e. 'phasic', which decayed during a maintained depolarization step, and 'tonic' which constituted the sustained elevation of [Ca2+]i above resting level. 3. A short (1 s) application of 10 mM caffeine mimicked the phasic component. After wash-out of caffeine, the subsequent depolarization induced a [Ca2+]i transient with reduced peak, the degree of suppression depending on the interval between wash-out of caffeine and depolarization. The phasic component of the depolarization and the caffeine-induced [Ca2+]i transients were not additive but saturative. 4. The phasic component was largely abolished in the continuous presence of 10 mM caffeine. It was also abolished by a 10 min cell dialysis of 10 microM ryanodine from the pipette solution and was strongly reduced by dialysis of 5 microM thapsigargin. Changes of the tonic component of the depolarization-induced [Ca2+]i transient were much less pronounced with all three interventions. 5. The tonic component of the depolarization-induced [Ca2+]i transient was increased when [Ca2+]o was elevated briefly before a depolarization close to 0 mV, whereas the phasic component was not significantly changed. Similarly, brief application of 1 microM Bay K 8644 increased the tonic component several-fold without modifying significantly the phasic component. 6. It is concluded that depolarization-induced influx of Ca2+ through L-type Ca2+ channels induces the release of Ca2+ from intracellular caffeine-sensitive stores which constitutes the major part of the phasic component. Ca2+ release superimposes on the effects of Ca2+ influx through L-type Ca2+ channels, the non-inactivating part of which constitutes the tonic component of the [Ca2+]i transient. Since the two processes interact, a dissection by simple subtraction is not possible.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Kinetic determination of talin-actin binding.

Smooth muscle talin prepared from chicken gizzard binds to skeletal muscle actin in vitro. The stoichiometry of 1:3 for talin:fluorescent labelled G-actin was confirmed by steady state titration and viscosity measurements under non-polymerizing conditions. The binding constant (Kd) of talin and G-actin was determined by continuous fluorescence titration and gave a value of approx 0.3 microM. The association rate constant of talin and fluorescent labelled G-actin of approx 7 x 10(6) M-1 x s-1 was ascertained by the stopped flow method; the dissociation rate constant was calculated at approx 2-3 s-1.

Actins↗
PubMed1991

Talin binds to actin and promotes filament nucleation.

Platelet talin binds to actin in vitro and hence is an actin binding protein. By four different non-interfering assay conditions (fluorescence, fluorescence recovery after photobleaching, (FRAP), dynamic light scattering and DNase-I inhibition) we show that talin promotes filament nucleation, raises the filament number concentration and increases the net rate of actin polymerization but has no inhibitory effect on filament elongation. Binding of talin to actin occurs at a maximal molar ratio of 1:3 as determined by fluorescencetitration under G-buffer conditions. The overall binding constant was approximately 0.25 microM.

Actins↗
PubMed1991

Human platelet P-235, a talin-like actin binding protein, binds selectively to mixed lipid bilayers.

The interaction of platelet talin (P-235) with mixtures of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylserine (DMPS) as well as with pure lipids was studied in reconstituted lipid bilayers. Incorporation of platelet talin into vesicles was achieved by self-assembly during cycles of freeze-thawing of co-dispersions containing vesicles and the purified protein. The yield of protein incorporation as a function of lipid composition was determined by measuring the protein/lipid ratio using protein assay, phosphate determination and gel electrophoresis in parallel. Protein-lipid interactions are monitored by high sensitive differential scanning calorimetry (DSC) measuring (i) the shifts of transition states delta Ts* and delta Tl*, where Ts represents the solidus line, the onset of lipid chain melting, and Tl the liquidus line, the endpoint of chain melting, and (ii) the heats of transition. Cytoplasmic talin differs from a membrane bound form by its ability and mode of lipid interaction. The latter partially penetrates into the hydrophobic region of the bilayer, which renders a low incorporation rate even into neutral lipids. This interaction is greatly enhanced in the presence of charged lipids: a marked shift of Tl occurs due to a selective electrostatic interaction of the protein with the membrane surface. Evidence for a selective binding is also provided by Fourier transform infrared spectroscopy (FTIR). Right-side-out oriented platelet talin can be cleaved by proteinases, which truncate the extrinsic electrostatic binding domain but not the hydrophobic. In addition, reconstituted platelet talin, like in vivo, can be cleaved by thrombin. The interaction of cytoplasmic platelet talin with lipid bilayers is purely electrostatic. Our data suggest that protein reconstitution by freeze-thawing is an equilibrium process and that the protein distribution between the membrane and water is determined by the Nernst distribution law. Consequently, the work of protein transfer from water into the bilayer can be measured as a function of charged lipids.

Blood Proteins↗
PubMed1991

Carbon monoxide inhibits depolarization-induced Ca rise and increases cyclic GMP in visceral smooth muscle cells.

Monocytes were isolated from the urinary bladder of the guinea-pig. By means of the voltage clamp technique, whole cells were depolarized from -65 to +10 mV in order to increase the intracellular calcium concentration [Ca2+]i and to monitor this increase by means of the calcium activated potassium current IK.Ca. Superfusion of the cells with carbon monoxide-containing solutions for 2 min inhibited the signal to about 50% of the control suggesting depression of the depolarization-induced increase in [Ca2+]i. The CO-mediated inhibition of IK.Ca was partially reversed by wash-off of CO; flashes of high light intensity accelerated the rate of recovery. Sodium nitroprusside (0.01-1 mM) depressed the depolarization-induced increase in [Ca2+]i similar to CO. In multicellular preparations of the urinary bladder, CO-containing media were shown to increase the cGMP concentration by a factor of 2 in the absence and by a factor of 3 in the presence of 1 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). According to our previous work, CO binds to and activates soluble guanylate cyclase [Brüne B and Ullrich V, Mol Pharmacol 32: 497-504, 1987; Utz J and Ullrich V, Naunyn Schmiedebergs Arch Pharmacol 337 (Suppl): 299, 1988] and the rise in cGMP could thus effect [Ca2+]i by still unknown mechanisms.

Animals↗
PubMed1991

Large and rapid changes of myofibrillar total calcium during the cardiac cycle. Electron probe microanalysis of voltage-clamped guinea-pig ventricular myocytes.

At 36 degrees C and 2 mM [Ca2+]o, single guinea-pig ventricular myocytes were voltage clamped with patch electrodes. When paired pulsing had potentiated the contraction to the maximum, the cells were shock-frozen for electron probe microanalysis (EPMA). Shock-freezing was timed at the end of diastole (-80 mV) or at different times during systole (+5 mV). The same paired-pulse protocol was applied to another group of myocytes from which contraction was recorded and [Ca2+]i was estimated by microfluospectroscopy (50 microMNa-Indo-1). In potentiated cells, during the first pulse, contraction peaked within 128 +/- 25 ms after start of depolarization. [Ca2+]i peaked within 25 ms to 890 /+- 220 nM (mean +/- SEM) and fell within 100 ms to about 450 nM. sigma Camyo, the total calcium concentration in the overlapping myofilaments (A-band), was measured by EPMA in 17 potentiated myocytes. During diastole, sigma Camyo was 2.6 +/- 0.4 mmol/kg dry weight (dw), which can be converted to 0.65 mM (mmoles per liter myofibrillar space). Since [Ca2+]i was 180 nM, we estimate that 99.97% of total calcium is bound. A time-course for systolic sigma Camyo was determined by shock-freezing 13 cells at different times after start of depolarization to +5 mV. sigma Camyo was 5.5 +/- 0.3 mmol/kg dw (1.4 mM) after 15-25 ms, 4.6 +/- 0.5 mmol/kg dw (1.1 mM) after 30-45 ms, and 3.1 mmol/kg dw (0.8 mM) after 60-120 ms. The fast time-course of sigma Camyo suggests that calcium binds to and unbinds from troponin C at a fast rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Endothelin depolarizes myocytes from porcine coronary and human mesenteric arteries through a Ca-activated chloride current.

The effect of endothelin (ET) on membrane potential and current was studied in myocytes isolated from porcine coronary or from human mesenteric arteries at 3.6 mM extracellular Ca2+ concentration and 37 degrees C. ET (1-100 nM) induced cell shortening and membrane depolarization from a resting potential of -50 mV to about -15 mV. Ca currents (ICa, L-type) were transiently reduced by ET. At -50 mV, ET induced an inward current that peaked within 2 s and fell within 10 s to a sustained level. The current could be enlarged by reducing bath extracellular Cl- ion concentration, but removal of extracellular Na+ ions had no effect. The voltage dependence suggests that the ET-induced current is a Cl current (ICl) at potentials negative to -30 mV; at more positive potentials K currents (IK,Ca) are superimposed. The effects of ET on ICa, ICl, IK,Ca and contraction were prevented by intracellular Ca chelators, suggesting a Ca-dependent activation mechanism. The ET effects were abolished by pretreatment with 20 mM caffeine or prior cell-dialysis with heparin [thought to block inositol triphosphate-induced sarcoplasmic reticular Ca release]. The results suggest that ET releases Ca from the SR through a phosphoinositol response and that the released Ca acts as second messenger in modulating the membrane currents.

Animals↗
PubMed1991

Depolarization-mediated intracellular calcium transients in isolated smooth muscle cells of guinea-pig urinary bladder.

1. Free intracellular calcium concentration ([Ca2+]i) was recorded in single smooth muscle cells of the guinea-pig urinary bladder held under voltage clamp at 36 degrees C and 3.6 mM-extracellular Ca2+. The Ca2+ indicator Indo-1 was loaded into the cells through patch electrodes. To separate Ca2+ currents (ICa), superimposed K+ currents were suppressed with a Cs(+)-containing electrode solution. 2. At a holding potential of -60 mV, resting [Ca2+]i was 114 +/- 22 nM (mean +/- S.D.). During 160 ms depolarization steps to 0 mV, [Ca2+]i rose to 885 +/- 140 nM. With steps of varied duration, peak [Ca2+]i increased with the time of depolarization up to about 1 s. Upon repolarization [Ca2+]i recovered to resting levels with a half-decay time of about 1 s; recovery was not significantly changed with repolarization potentials between -50 and -100 mV. 3. The potential dependence of the above peak [Ca2+]i transients was bell shaped, with a threshold around -40 mV and a maximum at 0 mV. During depolarization steps to potentials more positive than +80 mV [Ca2+]i did not significantly rise. 4. During step depolarizations to 0 mV lasting 10 s or longer, [Ca2+]i peaked within 814 +/- 18 ms and then decayed to a sustained level of 250 +/- 60 nM. The amplitude of the [Ca2+]i peak as well as the time course of the transient depended on the amplitude of ICa. The depolarizations increased [Ca2+]i to a sustained level with no clearly defined peak when ICa was reduced by partial inactivation or during steps close to the threshold of ICa (-40 mV). 5. The sustained level of [Ca2+]i with longer depolarizations of several seconds showed a bell-shaped voltage dependence with a maximum close to 0 mV. A bell-shaped voltage dependence for [Ca2+]i was also found during ramp-like depolarizations. However, when the rate of depolarization was low (7.5 mV s-1), the peak [Ca2+]i was found at more negative potentials (-15 mV). 6. The results are compatible with the idea that Ca2+ influx through voltage-operated Ca2+ channels is the key event in depolarization-mediated changes in [Ca2+]i in smooth muscle cells from urinary bladder.

Animals↗
PubMed1991

Total and free myoplasmic calcium during a contraction cycle: x-ray microanalysis in guinea-pig ventricular myocytes.

1. At 36 degrees C and 2 mM [Ca2+]o single guinea-pig ventricular myocytes were voltage clamped with patch electrodes. With a paired-pulse protocol applied at 1 Hz, a first pulse to +5 mV was followed by a second pulse to +50 mV. When paired pulsing had potentiated the contraction to the maximum, the cells were shock-frozen for electron-probe microanalysis (EPMA). Shock-freezing was timed at the end of diastole (-80 mV) or at different times during systole (+5 mV). 2. The same paired-pulse protocol was applied to another group of myocytes from which contraction and [Ca2+]i was estimated by microfluospectroscopy (50 microM-Na5-Indo-1). Potentiation moderately reduced diastolic sarcomere length from 1.85 to 1.82 microns and increased diastolic [Ca2+]i from about 95 to 180 nM. In potentiated cells, during the first pulse, contraction peaked within 128 +/- 25 ms after start of depolarization. [Ca2+]i peaked within 25 ms to 890 +/- 220 nM (mean +/- S.E.M.) and fell within 100 ms to about 450 nM. 3. Sigma Camyo, the total calcium concentration in the overlapping myofilaments (A-band), was measured by EPMA in seventeen potentiated myocytes. During diastole, sigma Camyo was 2.6 +/- 0.4 mmol (kg dry weight (DW]-1 which can be converted to 0.65 mM (mmoles per litre myofibrillar space). Since [Ca2+]i was 180 nM, we estimate that 99.97% of total calcium is bound. 4. A time course for systolic sigma Camyo was determined by shock-freezing thirteen cells at different times after start of depolarization to +5 mV. Sigma Camyo was 5.5 +/- 0.3 mmol (kg DW)-1 (1.4 mM) after 15-25 ms, 4.6 +/- 0.5 mmol (kg DW)-1 (1.1 mM) after 30-45 ms, and 3.1 mmol (kg DW)-1 (0.8 mM) after 60-120 ms. The fast time course of sigma Camyo suggests that calcium binds to and unbinds from troponin C at a fast rate. Hence, it is the slow kinetics of the cross-bridges that determines the 130 ms time-to-peak shortening. 5. Mitochondria of potentiated cells contained during diastole a total calcium concentration, sigma Camito, of 1.3 +/- 0.2 mmol (kg DW)-1 (0.4 mM). During the initial 15-25 ms of systole, sigma Camito did not change, however, during 30-45 ms sigma Camito rose to 3.7 +/- 0.5 mmol (kg DW)-1 (1.2 mM). The data suggest that sigma Camito can follow sigma Camyo with some delay, thereby participating in both slow diastolic and fast systolic changes in total calcium (sigma Ca), at least under the given conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Ca2+ influx through ATP-gated channels increments [Ca2+]i and inactivates ICa in myocytes from guinea-pig urinary bladder.

1. Whole-cell patch clamp was combined with microspectrofluometry (Indo-1) to study the effects of bath applied ATP on membrane currents and cytoplasmic Ca2+ concentration ([Ca2+]i) in single smooth muscle cells of the guinea-pig urinary bladder. Experiments were carried out at 22 degrees C and in 3.6 mM [Ca2+]o. Superimposed K+ currents were reduced by Cs+ dialysis from the patch electrode. 2. At -60 mV, ATP induced an inward current (Ins,ATP) that peaked within 0.4 s and then decayed. Ins,ATP was activated half-maximally by 1.1 microM-ATP and saturated at 50 microM-ATP to -1.1 +/- 0.2 nA (mean +/- S.E.M.). At 3.6 mM [Ca2+]o, Ins,ATP had a reversal potential (Erev) of -5 +/- 2 mV. From the shifts in Erev during changes in [Na+]o or [Ca2+]o we estimated that approximately 7% of Ins,ATP is carried by Ca2+ ions. 3. ATP (50 microM) increased [Ca2+]i transiently from resting 130 +/- 40 nM to 730 +/- 100 nM. At 22 degrees C, [Ca2+]i rose at a rate proportional to the instantaneous current amplitude of Ins,ATP. This relation was lost, however, after warming to 36 degrees C which increased the peak Ins,ATP (Q10 = 1.25) but reduced the peak of the ATP induced [Ca2+]i transient (Q10 = 0.75). We suggest that warming to 36 degrees C stimulated Ca2+ sequestration and Ca2+ efflux to such a degree that peak [Ca2+]i was attenuated significantly. 4. The contribution of Ca2+ ions to Ins,ATP was evaluated from a comparison of the increments in [Ca2+]i due to Ins,ATP and due to L-type Ca2+ channel current (ICa). For the same increment, Ins,ATP had to transport 19 times more charge than ICa. This number suggests that 5.8 +/- 0.8% of Ins,ATP is carried by Ca2+ ions which can be translated into a permeability ratio of PNa:PCa approximately 1:1. 5. During bath application of ATP, peak ICa was inhibited by 80 +/- 15%. Inhibition of ICa diminished to 20 +/- 8% after cell dialysis with 40 mM-EGTA, and it was 19 +/- 7% when extracellular Ca2+ had been substituted by Ba2+. These results are in agreement with the hypothesis of 'ICa inactivation by Ca2+'. Depletion of intracellular Ca2+ stores by pre-treatment with 20 mM-caffeine did not attenuate significantly the ATP-induced rise in [Ca2+]i or the ATP-induced inhibition of ICa. 6. The ATP-induced [Ca2+]i transients and the reduction of peak ICa recovered along a similar time course.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1991

Stimulation-induced potentiation of T-type Ca2+ channel currents in myocytes from guinea-pig coronary artery.

1. Whole-cell Ca2+ channel currents were studied in myocytes isolated from guinea-pig circumflex coronary artery at 36 degrees C and with 10 mM-Ba2+ (or Ca2+) as charge carrier. With 180 ms clamp steps from the holding potential of -100 mV, currents at -30 mV were carried mostly through the T-type calcium channels while at positive potentials currents were mostly of the L-type. 2. The increase in frequency of pulsing from 0.1 to 2.5 Hz resulted in a reduction of peak inward current ('negative staircase') with the 180 ms pulses to + 10 mV, but in a 2-fold potentiation ('positive staircase') with pulses to -30 mV. T-type currents and their frequency-mediated potentiation did not change significantly when Ba2+ was substituted by Ca2+ or Sr2+. 3. Potentiation of T-type currents was further analysed with a paired-pulse protocol: at a basal frequency of 0.1 Hz, a pre-pulse (inducing current I1) was followed by a 200 ms repolarization to -100 mV and a test pulse (inducing current I2). The potentiation could only be recorded using test pulses depolarizing the membrane to potentials between -40 and -10 mV; at more positive test potentials it was masked by the depressant effect of pre-pulses on the L-type current. 4. Potentiation of I2 by 200 ms pre-pulses started at pre-pulse potentials more positive than -60 mV and saturated at -20 mV (I2 potentiated by a factor 2.4). Between -20 and +130 mV the potentiation was not dependent on the pre-pulse potential suggesting that the influx of Ba2+ or Ca2+ is not required for this effect. Potentiation of I2 by a 10 s pre-pulse followed the voltage dependence of the steady-state inactivation curve of the T-type Ca2+ channel; potentiation became visible at potentials more positive than -80 mV and saturated at about -50 mV. 5. When changing the interval between two identical 200 ms pulses, the T-type current was found to recover completely from inactivation within 40 ms at -100 mV; at intervals of 160-320 ms maximal potentiation of I2 occurred. 6. With pre-pulses shorter than 200 ms, potentiation became attenuated when inactivation became less complete. When the potential during the interval between the pulses was -80 instead of -100 mV, maximal potentiation was reduced (I2 potentiated by a factor of 1.3 instead of 2.2) and occurred later (1.28 s). 7. Potentiated T-type currents inactivated faster.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Myocytes isolated from porcine coronary arteries: reduction of currents through L-type Ca-channels by verapamil-type Ca-antagonists.

Myocytes were enzymatically isolated from large epicardial arteries of the pig. In the cell attached configuration, we studied currents through L-type Ca-channels. At 22 degrees C, open channel conductance was 9 pS with 110 mM Ca2+ and 24 pS with 110 mM Ba2+ as charge carrier. According to the life time of the open state, 2 'modes' of gating are distinguished; mode 1 contributed time constants shorter than 1 ms, mode 2 those longer than 6 ms to the open time distribution. Mode 2 openings appeared spontaneously, more frequently with Ba2+ than with Ca2+ as charge carrier. The Ca-agonist Bay K 8644 (0.5 microM) facilitated the appearance of mode 2. Bath application of the phenylalkylamine D600 (1 microM) did not change the gating modes, but it reduced the channel openness by increasing the percentage of blank records. With whole cell recordings, we studied reduction of ICa by 1 microM D 600 at 3.6 mM [Ca2+] and 35 degrees C. At a holding potential of -45 mV, D 600 induced an 'initial block' of 35% (10% at -65 mV). Upon repetitive 1 Hz pulsing (170 ms to 0 mV) an additional, 'use-dependent' block developed with time. More negative holding potentials attenuated reduction of ICa by D 600, hyperpolarizations to -100 mV had an 'unblocking' effect. In regard to reduction of ICa, we compared the partially uncharged D 600 (membrane permeable) with the completely charged compound D 890 (membrane impermeable). When applied with the bath, 1 or 10 microM D 600 reduced ICa dose-dependently whereas D 890 was ineffective. When D 890 was applied via the patch electrode to the cytosol, it reduced ICa. We discuss that D 600 enters the cell in the uncharged lipid soluble form and reaches form the inside its receptor associated with the Ca-channel.

Animals↗
PubMed1990

L-type Ca-channels: similar Q10 of Ca-, Ba- and Na-conductance points to the importance of ion-channel interaction.

The temperature-dependence of currents through L-type Ca-channels was studied in myocytes isolated from the urinary bladder of the guinea pig. Currents were measured at 22 degrees C and 35 degrees C with Ca-, Ba- and Na-ions as charge carrier. The higher temperature increased the open channel conductance for Ca-ions from 8.5 to 16 pS (Q10 = 1.63 +/- 0.07, mean +/- S.D.), for Ba-ions from 24 to 43 pS (Q10 = 1.55 +/- 0.06), and for Na-ions (pH 9) from 74 to 131 pS (Q10 of 1.55 +/- 0.09). The differences in the Q10's are not significant, the activation energy approximates a common high value of 34.8 +/- 2.5 kJ/mol. A three barrier model with intra-channel binding predicts high Q10's for Ca and Ba but not for Na. To fit the results we postulate that the temperature-dependence reflects multiple ion-channel interactions within a central permeability barrier, e.g. polar groups substituting part of the ionic water shell.

Animals↗
PubMed1990

A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor.

Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross-linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays.

Actins↗