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W Trautwein

Publications and source records attributed to W Trautwein.

At least 55 records · Page 3Linked to original sources
PubMed1988

Modification of L-type calcium current by intracellularly applied trypsin in guinea-pig ventricular myocytes.

1. The L-type Ca2+ current was recorded in guinea-pig ventricular myocytes by the patch clamp technique in the whole-cell configuration. The modification of the current by intracellular application of proteases was studied. 2. During the first phase of action, trypsin, an endopeptidase, increased the amplitude of Ca2+ current about 3-fold. 3. Thereafter, there was a drastic slowing of the inactivation time course of the enhanced Ca2+ current. The half-time of inactivation increased from a control value of about 25 ms to values larger than 200 ms. 4. Cell dialysis with carboxypeptidase A, an exopeptidase, also enlarged the amplitude of Ca2+ current, but did not affect the kinetics of Ca2+ current. Leuaminopeptidase did not modify the Ca2+ current. 5. The hypothesis that Ca2+ channels are affected by the protease is supported by the fact that alterations of the extracellular Na+ or K+ concentration did not influence the modification of the membrane current. Another argument for the involvement of Ca2+ channels is that the modified membrane current could be blocked by inorganic and organic Ca2+ channel blockers (e.g. 10 microM-Cd2+, 100 microM-La3+ or 1 microM-D600). 6. Although the actions of trypsin and maximal concentrations of isoprenaline on the amplitude of the Ca2+ current were not additive, the slowing of inactivation by trypsin occurred independently from beta-adrenergic stimulation. 7. The effect of trypsin on the Ca2+ current could not be blocked by intracellular 5'-adenylyl-imidodiphosphate (AMP-PNP) or Rp-adenosine 3'5'-monothionophosphate (Rp-cAMPS), both of which are known to suppress the cyclic AMP-dependent phosphorylation of the Ca2+ channel. 8. It was concluded that trypsin may directly modify the membrane protein which forms the Ca2+ channel. Since the increment in peak Ca2+ current resembled the action of cyclic AMP-dependent phosphorylation, it may be related to the removal of a 'chemical' inactivation gate which is normally controlled by phosphorylation. The slowing of the time course of Ca2+ current inactivation by trypsin could be due to a modification of the voltage-dependent inactivation gate. Alternatively, the endopeptidase might remove an internal Ca2+ binding site normally responsible for Ca2+-dependent inactivation.

Adenylyl Imidodiphosphate↗
PubMed1987

Currents through ionic channels in multicellular cardiac tissue and single heart cells.

Ionic channels are elementary excitable elements in the cell membranes of heart and other tissues. They produce and transduce electrical signals. After decades of trouble with quantitative interpretation of voltage-clamp data from multicellular heart tissue, due to its morphological complexness and methodological limitations, cardiac electrophysiologists have developed new techniques for better control of membrane potential and of the ionic and metabolic environment on both sides of the plasma membrane, by the use of single heart cells. Direct recordings of the behavior of single ionic channels have become possible by using the patch-clamp technique, which was developed simultaneously. Biochemists have made excellent progress in purifying and characterizing ionic channel proteins, and there has been initial success in reconstituting some partially purified channels into lipid bilayers, where their function can be studied.

Animals↗
PubMed1987

Regulation of the cardiac calcium channel by protein phosphatases.

The calcium current (ICa) through the L-type channel in cardiac ventricular cells is enhanced by phosphorylation of a channel protein [Kameyama, M., Hofmann, F. & Trautwein, W. (1985) Pflügers Arch. Eur. J. Physiol. 405, 285-293]. We investigated the possible contribution of the 'catalytic subunits' of protein phosphatase 1 and 2A in the down-regulation of the cardiac calcium channel. Single guinea-pig ventricular myocytes were voltage clamped and the following results were obtained. (1) Intracellular perfusion of the myocyte with the catalytic subunits of protein phosphatase 1 (2 microM) as well as 2A (2.3 microM) completely abolished the increase of ICa induced by isoprenaline (0.05 microM) but did not decrease the basal level of ICa. Alkaline and acid phosphatases were without detectable effect. (2) Cell dialysis with the modulator of protein phosphatase 1 (inhibitor-2) under control conditions (without addition of isoprenaline) caused a slow significant increase of ICa. (3) The time course for the wash-out of the isoprenaline effect was considerably prolonged in the presence of high concentrations of inhibitor-2. (4) Perfusion of the myocyte under basal conditions with adenosine 5'-[gamma-thio]triphosphate led to a slow increase of ICa. Additional superfusion of the cell with a threshold concentration of isoprenaline (0.01 microM) resulted in a rapid increase of ICa which could not be washed out during at least 10 min. From these results we make the following conclusions. (1) The calcium channel from guinea-pig myocytes is regulated by phosphorylation-dephosphorylation. (2) The catalytic subunits of the protein phosphatases 1 as well as 2A, purified from rabbit skeletal muscle, catalyse the down-regulation of the channel. (3) Indirect evidence suggests that endogenous protein phosphatase 1 contributes only partially to the dephosphorylation of the calcium channel in the intact myocyte.

Animals↗
PubMed1987

On the relationship between V max of slow responses and Ca-current availability in whole-cell clamped guinea pig heart cells.

The relationship between Ca current availability and maximum rate of rise (V max) of slow responses was determined in the same single guinea pig ventricular heart cell under voltage and current clamp conditions (whole-cell clamp technique). The results are as follows. (1) Cell capacitance measured in 32 cells from the current response to a fast ramp voltage-clamp pulse (119.6 +/- 4.6 pF, mean +/- SE) or from Vmax values at a holding potential of -50 or -40 mV (118.6 +/- 5.3 pF) are identical. (2) In control conditions ([Ca]o 1.8, [K]o 4 and [Cs]i 140 mM), voltage-dependence of steady-state inactivation of Ca current (ICa) or Vmax are similar up to -35 mV. However, Vmax significantly (P less than 0.005) underestimates ICa availability at more positive potentials. At -30 mV, ICa and Vmax amplitudes represent respectively 35.6 and 22.4% (n = 14) of their maximum value. (3) In the presence of 50 nM isoprenaline, Vmax and the underlying ICa are respectively increased by 79.2 +/- 13.8% and 71.2 +/- 13.8% (n = 15). No statistically significant deviation from linearity is then observed. (4) When Vmax amplitude is expressed as a function of ICa density, an almost linear relationship is observed for Vmax values between 0 and 25 V/s. Vmax is then best described by the equation: Vmax (V/s) = 1.043 ICa (pA/pF) -0.514 (46 cells). (5) We conclude that, under conditions that minimize outward currents, Vmax of slow responses accurately measures ICa amplitude, except when ICa is decreased to less than 40% of its maximum control amplitude (i.e., below 4 pA/pF). At that point, Vmax underestimates ICa.

Action Potentials↗
PubMed1987

On the mechanism of histamine induced enhancement of the cardiac Ca2+ current.

In guinea pig ventricular myocytes, the effect of histamine on the slow Ca2+ current (ICa) was studied and the following results were obtained: (1) Superfusion of cells with histamine resulted in a dose-dependent enhancement of the amplitude of ICa. The threshold concentration of histamine was 10(-8) M, half maximal increase occurred at 3 X 10(-7) M and maximal enhancement (about 3-4-fold) at 5 X 10(-6) M. (2) The histamine effect was greatly reduced by the H2 antagonist cimetidine (10(-5) M) but only slightly by the H1 antagonist diphenhydramine (10(-5) M). (3) Effects of isoprenaline (ISP) and histamine at maximal effective concentrations on ICa were not additive, suggesting that both agents use the same intracellular pathway. Intracellular infusion of a blocker of the cAMP-dependent protein kinase, Rp-cAMPS (10(-4) M), prevented the histamine effect. (4) The involvement of GTP-dependent transducer proteins was studied by cell dialysis with several GTP derivatives. Intracellular application of the stable GDP-analogue, GDP-beta-S, reduced the histamine effect on ICa, whereas the stable GTP analogue, GTP-gamma-S, mimicked the histamine effect.

Animals↗
PubMed1987

Modulation of calcium channel function by phosphorylation in guinea pig ventricular cells and phospholipid bilayer membranes.

L-type calcium channel activity of some excitable cells is markedly enhanced by beta-adrenergic agents. The enzymatic cascade underlying this important modulatory effect has been studied with patch-clamp techniques in single dialyzed ventricular cells from guinea pig heart. The steps between the binding of agonist to the beta-receptor and the increase in calcium influx can be summarized as follows: Agonist binding to beta-receptor greater than adenylate cyclase increases greater than cAMP increases greater than cA-kinase increases greater than protein phosphorylation greater than altered calcium channel properties greater than ICa increases A basal phosphorylation reaction seems not to be a prerequisite for calcium channel function. By combining molecular and functional approaches, the purified dihydropyridine-receptor complex from rabbit skeletal muscle transverse-tubules can be reconstituted in phospholipid bilayer membranes to form a functional 20-pS calcium channel that retains the principal regulatory, biochemical, and pharmacologic properties of membrane-bound L-type calcium channels.

Animals↗
PubMed1986

Modulation of Ca current during the phosphorylation cycle in the guinea pig heart.

The calcium current (ICa) in the heart is increased by phosphorylation of a protein which is part of, or close to, the Ca channel. The phosphorylation is catalysed by cAMP-dependent protein kinase (cAMP-PK). The question whether dephosphorylated channels are available to open on depolarization was examined in ventricular myocytes of guinea pig by recording whole cell ICa during dialysis with either regulatory (R) subunit of cAMP-PK or protein kinase inhibitor (PKI) or adenosine-5'-(gamma-thio)-triphosphate (ATP gamma S). The following results were obtained: 1) R subunit reduced and PKI reversed the isoprenaline (ISP)-induced enhancement of ICa, suggesting their ability to inhibit cAMP-PK. 2) R subunit and PKI, however, reduced basal (i.e. non beta-adrenergically stimulated) ICa only by about 20%. 3) Dialysis with ATP gamma S resulted in a slow increase in basal ICa, presumably due to dephosphorylation-resistant thiophosphorylation. 4) When, however, the cell was dialyzed with PKI the effect of ATP gamma S was almost completely suppressed, suggesting no detectable phosphorylation related to the channel activity in this condition. These results support the view that even in the dephosphorylated state Ca channels are available to open on depolarization and that phosphorylation by cAMP-PK increases the opening probability.

Adenosine Triphosphate↗
PubMed1986

On the mechanism of muscarinic inhibition of the cardiac Ca current.

The mechanism of muscarinic inhibition of the Ca-current (ICa) was studied in ventricular myocytes of guinea pig hearts and the following results were obtained. Acetylcholine (ACh) in concentrations up to 10(-4) M had little effect, if any, on ICa in control cells. ACh reduced the isoprenaline (ISP)-induced increase of ICa. The dose-response-relation (ISP concentration vs. ICa density) was shifted by ACh towards higher ISP concentrations. But both, at low and high ISP concentrations ACh had nor or little effect. ACh was ineffective when ICa was increased by dialysing the cell with catalytic subunit of cAMP-dependent protein kinase or cAMP. ACh reduced ICa enhanced by isobutylmethylxanthine or by forskolin. ACh did not depress ICa when the cell was dialysed with the non-hydrolysable GTP-derivative, GMP-PNP. In this condition the beta-adrenergic enhancement of ICa was also absent. Pertussis toxin, which is known to inhibit the inhibitory transducer protein (Ni), abolished the ACh response. We concluded from these results that ACh depresses ICa by inhibiting, via Ni, the cAMP production.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Voltage-dependent properties of macroscopic and elementary calcium channel currents in guinea pig ventricular myocytes.

Whole-cell Ca channel currents were recorded from guinea pig ventricular myocytes that were internally perfused with Cs solution and bathed in solutions containing 3.6 mM Ca, 3.6 mM Ba or 90 mM Ba (34 degrees C). Single Ca channel currents were recorded from cell-attached membrane patches of similar myocytes; the patch pipettes contained a 90 mM Ba solution. 1. Although the shape of the whole-cell I-V relation was independent of the bathing solution, this was not the case with the location of the inward current maximum (Vpeak); Vpeak in 90 mM Ba was about 30 mV positive to Vpeak in 3.6 mM Ba. 2. The activation and inactivation of whole-cell currents were voltage dependent. Compared to the voltage dependencies in 3.6 mM Ba, those in 90 mM Ba were shifted by about 30 mV to the right, suggesting a neutralization of surface charges. 3. Observations compatible with the ion permeation model proposed by Hess and Tsien (1984) included (a) a depression of current during Ca/Ba solution exchange, (b) a high divalent to monovalent ion permeability, and (c) rectification of the outward limb of the I-V relation. 4. Estimated current densities at Vpeak were similar for myocytes in 3.6 mM Ca and 3.6 mM Ba, and about 10 times larger in 90 mM Ba. 5. Average currents (I) calculated from ensembles of records of single Ca channel current had voltage-dependent time courses resembling those of whole-cell IBa (90 mM). 6. Single-channel I-V relations were superimposable on whole-cell I-V curves suggesting that voltage-dependent single-channel parameters (probability of opening, elementary current amplitude) can be related to the voltage-dependent macroscopic current parameters (activation, instantaneous I-V relation) when scaled by channel number. 7. The density of Ca channels in myocytes was calculated from whole-cell IBa (90 mM) and average current through single channels. The outcome, 3-5 channels/micron 2, agrees with two other recent estimates (Tsien et al. 1983; Lux and Brown 1984). However, it is difficult to reconcile with the much lower density that one would forecast from the frequency of functional channel observation in myocyte membrane patches (Pelzer et al. 1985c).

Animals↗
PubMed1986

Fast and slow gating behaviour of single calcium channels in cardiac cells. Relation to activation and inactivation of calcium-channel current.

The Ca-channel gating behaviour during steady and stepwise depolarization was examined in recordings of single Ca-channel activity from cell-attached membrane patches of single ventricular cells isolated enzymatically from hearts of adult guinea pigs. The single-channel recordings were performed by means of the improved patch-clamp technique (Hamill et al. 1981) with 90 mM Ba in the pipettes. Upon step depolarization, two types of current records were regularly observed in the ensembles: (1) traces with Ca-channel activity (in the form of closely-spaced brief pulses of inward current with a unitary amplitude) of various length, and (2) blank sweeps without any detectable single-channel opening. The records with Ca-channel activity show a distinct tendency for openings to occur towards the beginning of the clamp pulse, followed by long periods of silence. The blank sweeps seem to reflect a condition or conditions where the Ca channel is unavailable for opening. The corresponding ensemble mean current I(t) displayed a rapid rising phase to its peak followed by a slow decay. During steady depolarization, kinetic analysis of the distributions of all open and shut lifetimes revealed a monoexponential probability density distribution function of all open times. By contrast, more than two exponential terms were required for an accurate description of the frequency distribution of all shut lifetimes. Corresponding to the two well-separated fast closed time components, individual Ca-channel openings were grouped into bursts of openings. The bursting behaviour reflected fast gating transitions and was related to the fluctuations of the Ca channel between two short-lived closed states and one open state. This fast gating was terminated by the entrance of the Ca channel into at least one long-lived closed state, exit from which was slow in comparison to the rapid cycling. As consequence, bursts of openings were further grouped together in clusters of bursts, the cluster behaviour being related to slow gating transitions in the kinetics of the Ca channel. The biphasic frequency distribution of the first latencies (resulting from the transit through the two short-lived shut states, before the open state is entered) superimposed on the first time derivative of the rising phase of the ensemble mean current, I(t), upon step depolarization. The time constant of the monoexponential distribution function of all cluster lifetimes matched the declining phase of I(t) during maintained depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

The protein-specific phosphatase 1 antagonizes the beta-adrenergic increase of the cardiac Ca current.

In isolated ventricular cells from the adult guinea pig heart the slow Ca current was recorded during beta-adrenergic stimulation and during cell dialysis with a protein-specific phosphatase-1 (PPase-1). The increase in the amplitude of ICa during bath application of isoprenaline (5 X 10(-8) M) could be completely reversed by dialysing the cell with 2 microM PPase-1. Lower enzyme concentrations produced smaller effects. The control amplitude of ICa was only little affected by dialysis with PPase-1. The result suggests that PPase-1 is a likely candidate for the downregulation of Ca channels, that Ca channels can open in the dephosphorylated state.

Animals↗
PubMed1986

Intracellular control of calcium and potassium currents in cardiac cells.

The intracellular mechanisms by which cardiac Ca current (ICa) and the delayed outward K current (IK) are modulated during beta-adrenergic or muscarinic stimulation were investigated at the level of both single-channel and whole-cell currents in single ventricular myocytes of guinea-pigs. Superfusion of cells with beta-adrenergic agonist increased the amplitude of whole-cell ICa in a dose-dependent manner. In the single-channel recording, neither the amplitude of elementary current nor the total number of active channels was affected but the number of blank records was markedly reduced resulting in a larger amplitude of the ensemble average current. Intracellular dialysis of cells with cyclic AMP (cAMP) or the catalytic (C) subunit of cAMP-dependent protein kinase (cAMP-PK) produced a dose-dependent increase in the amplitude of ICa and IK. A non-hydrolysable ATP analogue, AMP-PNP, reduced whereas ATP gamma S enhanced the effects of beta-agonist on ICa and IK, suggesting an involvement of protein phosphorylation during the enhancement of these currents. The regulatory subunit of cAMP-PK, the heat-stable protein-kinase inhibitor (PKI) and type-1 protein phosphatase antagonized the beta-adrenergic enhancement of ICa and IK, but did not eliminate ICa. Acetylcholine (ACh) reduced the amplitude of ICa when ICa was enhanced by either beta-adrenergic agonist, forskolin or 3-isobutyl-1-methyl-xanthine but did ACh not when ICa was enhanced by intracellular dialysis with cAMP or C subunit, suggesting that muscarinic inhibition occurs at the level of adenylate cyclase. Non-hydrolysable GTP analogue, GMP-PNP, uncoupled both beta-adrenergic and muscarinic modulation of ICa. Pertussis toxin selectively eliminated the effect of ACh on ICa. Based on these results, we concluded that the activities of the Ca channel and the delayed outward K channel are controlled by the action of neurotransmitters, which are mediated by GTP-binding proteins and cAMP-dependent protein phosphorylation. It is suggested that phosphorylation of 'Ca-channel-related protein' leads to an increased open probability without changing the total number of channels or the elementary current amplitude.

Adrenergic beta-Agonists↗
PubMed1985

On the mechanism of beta-adrenergic regulation of the Ca channel in the guinea-pig heart.

Dose-response relations for the increase in the amplitude of Ca current (ICa) on external application of isoprenaline (ISP) and internally applied cyclic AMP (cAMP) or catalytic subunit of cAMP-dependent protein kinase (C subunit) were established in single ventricular cells of the guinea pig. An intracellular dialysis technique was used. The threshold concentration was for ISP 10(-9) M, for cAMP 3 microM (pipette concentration to which 10(-5) M 3-isobutyl-1-methylxanthine was added) and for C subunit around 0.4 microM (pipette concentration). The concentrations for the half-maximal effect were 3.7 X 10(-8) M (ISP), 5.0 microM (cAMP) and 0.95 microM (C subunit) and for the maximum effect 10(-6) M (ISP), 15-20 microM (cAMP) and 3-4 microM (C subunit). For all three agents the maximum increase in the Ca current density was similar (a factor of 3-4), suggesting that they converge on the same site of the Ca channel. Accordingly, the effects of cAMP and C subunit on ICa were non-additive to those of ISP. From these data the relationship both between concentrations of ISP and cAMP and between those of cAMP and active C subunit in terms of their effects on ICa could be estimated and were compared with those obtained in broken cell preparations. A competitive inhibitor of phosphorylation, 5'-adenylyl-imidodiphosphate (5 mM), greatly reduced the effects of ISP and C subunit on ICa. Cell dialysis with 3 mM adenosine-5'-(gamma-thio)-triphosphate, which produces a dephosphorylation-resistant phosphorylation, markedly potentiated the effects of ISP and cAMP on ICa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Temperature-induced transitory and steady-state changes in the calcium current of guinea pig ventricular myocytes.

ICa was recorded in guinea pig ventricular myocytes using the whole-cell voltage-clamp technique. The shape of the I-V relation was unaffected by temperature (21-37 degrees C) but there were large changes in ICa amplitude and time course. Steady-state responses indicated Q10's of 2.96 +/- 0.14 (amplitude), 2.52 +/- 0.13 (time to peak), and 2.82 +/- 0.28 (T1/2 inactivation) (mean +/- SD, n = 6). Quick changes in temperature (T1/2 less than 30 s) induced pronounced deviations from the steady-state Q10 relations (early depression, compensatory overshoot). Thus, cardiac ICa differs from other currents in having a high amplitude-Q10 and an oscillatory response to rapid temperature changes.

Animals↗
PubMed1985

Cardiac calcium channels and their control by neurotransmitters and drugs.

Calcium channels, which play a primary role in the control of the calcium influx into cardiac cells, were initially studied by recording macroscopic currents in multicellular preparations. More recently, channel research has combined studies of whole cell calcium currents and elementary currents through single calcium channels, both measured in isolated cardiac cells. These studies provide insight into the mechanism of opening and closing of single calcium channels and enable inferences to be made about the whole cell calcium current from the average gating behavior of single channels. In addition, they promise a more complete understanding of the relation between the biophysical properties and molecular structure of the calcium channel. New information has also been obtained on the modulation of calcium channel gating by neurotransmitters and drugs.

Acetylcholine↗
PubMed1985

Cardiac Ca channel currents at the level of single cells and single channels.

Ca channels in excitable membranes are of great importance for many cellular functions. Modulation of these channels by voltage, neurotransmitters and drugs regulates Ca influx into the cell and thereby alters the functional state of the cell (7). For many years, quantitative analysis has been seriously complicated by experimental problems (see 2, 8, 10). There has been no preparation as suitable and no drug as specific for Ca channels, as the squid axon and TTX are for Na channels (10). The advent of the isolated adult heart cell and the development of powerful new electrophysiological methods, however, have opened up the possibility to record Ca channel activity at the level of single cells and single channels and to obtain evidence on molecular mechanisms of Ca channel gating and modulation.

Animals↗
PubMed1984

Beta-adrenergic increase in the calcium conductance of cardiac myocytes studied with the patch clamp.

In bovine, cat and guinea pig myocytes the effect of bath application of adrenaline or isoprenaline and of injection of cAMP on the Ca channel was studied with the patch clamp (Hamill et al. 1981), and the following results were obtained. On beta-adrenergic stimulation more activity of the single channel on repeated depolarizations and less records without activity were observed. Correspondingly, the average currents were increased. When the patch contained only one channel as judged from the lack of superpositions during all depolarizations, beta-adrenergic stimulation never produced superpositions indicating that the total number of channels did not increase. Also, it was never possible to activate a channel in the mute patch. The single channel conductance was not changed by catecholamines or cAMP. Increase in probability during depolarization of the channel to be in the open state was proven by non-stationary fluctuation analysis. The kinetic analysis showed a prolongation of the open times and shortening of the shut times by catecholamines, indicating that the rate constants in a three state model C1 in equilibrium C2 in equilibrium O are changed in such a way that the equilibrium shifted towards the open state. In some patches clusters of channels were observed, and activity was greatly increased by adrenaline, isoprenaline and cAMP-injection. The decay of the mean current was either mono-exponential or, in most cases, double-exponential. When the decay was mono-exponential, fluctuation analysis showed an increase in open probability on beta-stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗