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I Zahradník

Publications and source records attributed to I Zahradník.

At least 19 recordsLinked to original sources
PubMed2005

Computer-aided formation of the whole-cell patch-clamp recording configuration.

The conventional patch-clamp technique requires well-trained experimenter. Few commercial automated patch-clamp systems, designed for drug development, are better suited for large-scale research then for standard electrophysiological experiments. Here we describe a state machine for automated recognition of recording states of the patch-clamp experiment. The principle of the state machine is based on evaluation of the charge carried by membrane current during specific time segments in responses to square wave voltage stimulation. The state machine may serve for generating various sound alerts, signals for automated control of other devices, assistance in micromanipulation, internal pipette pressure control, and holding potential adjustments. Algorithm of the state machine, designed to cover wide variety of cell types, was successfully tested on rat ventricular myocytes.

Algorithms↗
PubMed2003

Regulation of dynamic behavior of cardiac ryanodine receptor by Mg2+ under simulated physiological conditions.

Mg2+, an important constituent of the intracellular milieu in cardiac myocytes, is known to inhibit ryanodine receptor (RyR) Ca2+ release channels by competing with Ca2+ at the cytosolic activation sites of the channel. However, the significance of this competition for local, dynamic Ca2+-signaling processes thought to govern cardiac excitation-contraction (EC) coupling remains largely unknown. In the present study, Ca2+ stimuli of different waveforms (i.e., sustained and brief) were generated by photolysis of the caged Ca2+ compound nitrophenyl (NP)-EGTA. The evoked RyR activity was measured in planar lipid bilayers in the presence of 0.6-1.3 mM free Mg2+ at the background of 3 mM total ATP in the presence or absence of 1 mM luminal Ca2+. Mg2+ dramatically slowed the rate of activation of RyRs in response to sustained (> or =10-ms) elevations in Ca2+ concentration. Paradoxically, Mg2+ had no measurable impact on the kinetics of the RyR response induced by physiologically relevant, brief (<1-ms) Ca2+ stimuli. Instead, the changes in activation rate observed with sustained stimuli were translated into a drastic reduction in the probability of responses. Luminal Ca2+ did not affect the peak open probability or the probability of responses to brief Ca2+ signals; however, it slowed the transition to steady state and increased the steady-state open probability of the channel. Our results indicate that Mg2+ is a critical physiological determinant of the dynamic behavior of the RyR channel, which is expected to profoundly influence the fidelity of coupling between L-type Ca2+ channels and RyRs in heart cells.

Animals↗
PubMed2003

Kinetics of cardiac RyR channel gating studied at high temporal resolution.

Measurements of ryanodine receptor (RyR) activity during dynamic changes of calcium concentration have suggested that RyR has at least four calcium binding sites, and that activation transpires as an increase in the activity within the high open probability H-mode. Binding of several Ca2+ ions within the H-mode should manifest itself in the steady-state RyR activity by the presence of multiple closed times. However, previously only two closed times were detected in the H-mode of RyR activity. Here we recorded steady-state activity of single cardiac RyRs with high temporal resolution and compared it to data simulated under the same conditions using our previously published model of RyR gating. At a 10 kHz resolution, the closed time histograms of both experimental and simulated data had three exponential components. The closed times of simulated data were not significantly different from those obtained experimentally. After filtering at 2 kHz, only two exponential closed time components with time constants not significantly different from those previously published could be detected in both experimental and simulated records. The conformity of the steady-state experimental data to the model derived from the dynamic data provides further support for the idea that RyRs need binding of multiple Ca2+ ions to open.

Animals↗
PubMed2003

Structure and composition of tubular aggregates of skeletal muscle fibres.

Unusual regions of densely packed membranous tubules known as tubular aggregates (TAs) have been observed in skeletal muscle fibres of mammals under numerous pathological conditions but also in health. Their causality is unclear. It is neither known whether TAs are destructive and should be treated or whether they have a compensating function in an endangered muscle. In spite of many similarities, the histochemical, immunocytochemical and ultrastructural characteristics of tubular aggregates do vary. Histochemistry provided an overall characteristic of TAs as membranous inclusions with a variety of enzymatic activities. Immunocytochemical evidence revealed that tubular aggregates contain miscellaneous proteins and that derive from membranes of sarcoplasmic reticulum and mitochondria. No evidence for the presence of contractile and cytoskeletal proteins in TAs was found. Ultrastructurally, TAs are characterized as more or less densely packed aggregates of vesicular or tubular membranes of variable forms and sizes that may contain amorphous material, filaments or inner tubules. Various reported types of tubular aggregates, namely, proliferating terminal cisterns, vesicular membrane collections, TAs with double-walled tubules, TAs with single-walled tubules, aggregates of dilated tubules with inner tubules, aggregates of tubulo-filamentous structures, filamentous tubules, riesentubuli, and related membranous structures including cylindrical spirals are sumarized and analyzed here in detail.

Animals↗
PubMed1999

Analysis of calcium-induced calcium release in cardiac sarcoplasmic reticulum vesicles using models derived from single-channel data.

The planar lipid bilayer and vesicle release experiments are two alternative approaches used to study the function of the ryanodine receptor (RyR) channel at the subcellular level. In this work, we combine models of gating (Zahradníková and Zahradník, Biophys. J. 71 (1996) 2996-3012) and permeation (Tinker et al., J. Gen. Physiol. 100 (1992) 495-517) of the cardiac RyR channel to simulate calcium release experiments on sarcoplasmic reticulum vesicles. The resulting model and real experimental data agreed well within the experimental scatter, confirming indistinguishable properties of the RyRC in the vesicle preparation and in the planar lipid bilayer. The previously observed differences in calcium dependencies of the release and the gating processes can be explained by binding of calcium within the RyRC conducting pore. A novel method of analysis of calcium dependence of calcium release was developed and tested. Three gating models of the RyRC, showing, respectively, an increase, no change, and a decrease in calcium sensitivity over time, were compared. The described method of analysis enabled determination of temporal changes in calcium sensitivity, giving potential for detection of the adaptation/inactivation phenomena of the RyRC in both vesicle and in situ release experiments.

Calcium↗
PubMed1999

Rapid activation of the cardiac ryanodine receptor by submillisecond calcium stimuli.

The local control concept of excitation-contraction coupling in the heart postulates that the activity of the sarcoplasmic reticulum ryanodine receptor channels (RyR) is controlled by Ca(2+) entry through adjoining sarcolemmal single dihydropyridine receptor channels (DHPRs). One unverified premise of this hypothesis is that the RyR must be fast enough to track the brief (<0.5 ms) Ca(2+) elevations accompanying single DHPR channel openings. To define the kinetic limits of effective trigger Ca(2+) signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca(2+) generated by flash photolysis of DM-nitrophen. Application of such Ca(2+) spikes (amplitude approximately 10-30 microM, duration approximately 0.1-0.4 ms) resulted in activation of the RyRs with a probability that increased steeply (apparent Hill slope approximately 2.5) with spike amplitude. The time constants of RyR activation were 0.07-0.27 ms, decreasing with spike amplitude. To fit the rising portion of the open probability, a single exponential function had to be raised to a power n approximately 3. We show that these data could be adequately described with a gating scheme incorporating four sequential Ca(2+)-sensitive closed states between the resting and the first open states. These results provide evidence that brief Ca(2+) triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings. They also provide evidence for the assumption that RyR activation requires binding of multiple Ca(2+) ions in accordance with the tetrameric organization of the channel protein.

Acetates↗
PubMed1996

A minimal gating model for the cardiac calcium release channel.

A Markovian model of the cardiac Ca release channel, based on experimental single-channel gating data, was constructed to understand the transient nature of Ca release. The rate constants for a minimal gating scheme with one Ca-free resting state, and with two open and three closed states with one bound Ca2+, were optimized to simulate the following experimental findings. In steady state the channel displays three modes of activity: inactivated 1 mode without openings, low-activity L mode with single openings, and high-activity H mode with bursts of openings. At the onset of a Ca2+ step, the channel first activates in H mode and then slowly relaxes to a mixture of all three modes, the distribution of which depends on the new Ca2+. The corresponding ensemble current shows rapid activation, which is followed by a slow partial inactivation. The transient reactivation of the channel (increment detection) in response to successive additions of Ca2+ is then explained by the model as a gradual recruitment of channels from the extant pool of channels in the resting state. For channels in a living cell, the model predicts a high level of peak activation, a high extent of inactivation, and rapid deactivation, which could underlie the observed characteristics of the elementary release events (calcium sparks).

Animals↗
PubMed1995

Description of modal gating of the cardiac calcium release channel in planar lipid membranes.

Single channel activity of the cardiac ryanodine-sensitive calcium-release channel in planar lipid membranes was studied in order to elucidate the calcium-dependent mechanism of its steady-state behavior. The single channel kinetics, observed with Cs+ as the charge carrier at different activating (cis) Ca2+ concentrations in the absence of ATP and Mg2+, were similar to earlier reports and were extended by analysis of channel modal behavior. The channel displayed three episodic levels of open probability defining three gating modes: H (high activity), L (low activity), and I (no activity). The large difference in open probabilities between the two active modes resulted from different bursting patterns and different proportions of two distinct channel open states. I-mode was without openings and can be regarded as the inactivated mode of the channel; L-mode was composed of short and sparse openings; and H-mode openings were longer and grouped into bursts. Modal gating may explain calcium-release channel adaptation (as transient prevalence of H-mode after Ca2+ binding) and the inhibitory effects of drugs (as stabilization of mode I), and it provides a basis for understanding the regulation of calcium release.

Adaptation, Physiological↗
PubMed1993

Multiple effects of caffeine on calcium current in rat ventricular myocytes.

Caffeine exerts a number of different effects on L-type calcium current in rat ventricular myocytes. These include: (1) a slowing of inactivation that is comparable to, but not additive to, that produced by prior treatment of the cells with ryanodine (a selective sarcoplasmic reticulum Ca2+ releaser) or high concentrations of intracellular 1,2-bis[2-aminophenoxy]ethane-N,N,N',-N'-tetraacetic acid (BAPTA) (a fast Ca2+ chelator), (2) a stimulation of peak ICa that is comparable to, but not additive to that produced by prior treatment with isobutylmethylxanthine (a selective phosphodiesterase inhibitor), and (3) a dose-dependent decrease of peak ICa that is not prevented by pretreatment with any of these agents. None of the caffeine actions could be mimicked or prevented by administration of 8-phenyltheophylline, a specific adenosine receptor antagonist. We conclude that only the slowing of ICa inactivation is due to caffeine's ability to deplete the sarcoplasmic reticulum of calcium. The stimulatory effect of caffeine on peak ICa is probably due to phosphodiesterase inhibition, while caffeine's inhibitory effect on ICa is independent of these processes and could be a direct effect on the channel. The multiplicity of caffeine actions independent of its effects on the sarcoplasmic reticulum lead to the conclusion that ryanodine, though slower acting and essentially irreversible, is a more selective agent than caffeine for probing sarcoplasmic reticulum function and its effects on other processes.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Modification of cardiac Ca2+ release channel gating by DIDS.

The effects of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on individual cardiac sarcoplasmic reticulum Ca2+ release channels have been examined in planar lipid bilayers. A sudden step-like increase in open probability (Po) induced by DIDS was observed. At zero holding potential it proceeded with an apparent time constant of 84 s and Po was increased 15 times in the steady state. Independent of membrane voltage, DIDS induced a long-lived open state with tau o = 15 ms at 0 mV and tau o = 5 ms at -50 mV (cis (cytoplasmic) side negative). While at 0 mV the modified channel spent most of the time in this long-lived state, at -50 mV the occupancy of the long-lived state was very low and most openings occurred to the short and medium open state, leading to deactivation of the channel to only 2-3 times of control. Voltage-induced changes in the activity of modified channels were reversible and proceeded with time constants of 14 s for deactivation and 12 s for activation, that is, faster than onset of activation by DIDS. As no changes in single channel conductance of the channel were observed in the presence of DIDS and its abrupt activating action could not be removed, a covalent modification of an amino group residing in the gating structure of the channel by DIDS can be considered.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Single potassium channels of human glioma cells.

Single potassium channels in the membrane of human malignant glioma cells U-118MG were studied using the technique of patch clamp in cell-attached and inside-out configurations. Three types of potassium channels were found which differed from each other under conditions close to physiological in their conductance and gating characteristics. The lowest-conductance channel (20 pS near the reversal potential) showed a mild outward rectification up to 45 pS at positive voltages and spontaneous modes of high and low activity. At extreme values of potentials its activity was generally low. The intermediate conductance channel had an S-shaped I-V curve, giving a conductance of 63 pS at reversal, and a low and voltage independent opening probability. The high-conductance (215 pS) channel was found to be activated by both membrane potential and Ca2+ ions and blocked by internal sodium at high voltages. The current-voltage curves of all three channel types displayed saturation.

Calcium↗
PubMed1992

Interaction of diltiazem with single L-type calcium channels in guinea-pig ventricular myocytes.

The effects of diltiazem on cardiac L-type calcium channels were studied at the single channel level, using Ba2+ ions as the charge carrier. Patch clamp experiments were performed on enzymatically isolated guinea-pig ventricular myocytes. It was shown in cell-attached configuration on multichannel patches that diltiazem, when applied to the bath, can approach the calcium channel under the pipette after diffusion through the membrane phase. The time constant of the onset of the effect was 60 s. The rate of recovery seemed to be of the same order. Diltiazem had most prominent effect on calcium channel open state probability by reducing the frequency of openings, and by increasing the frequency of records without channel opening (nulls). The effect on mean open time was found to be insignificant at 1 kHz resolution. Diltiazem had no effect on the amplitude of unitary currents. These data are consistent with the assumption that diltiazem interacts mainly with the inactivated state (although interaction with the closed states was not ruled out), and does not bind to the open state of the calcium channel.

Animals↗
PubMed1990

[Rubidium permeability through Ca2(+)-activated potassium channels in human erythrocytes].

Both potassium and rubidium can flow through the Ca2(+)-activated K channel of human erythrocytes. Although rubidium unitary currents are much smaller than potassium ones at the same potentials, no change in the reversal potential was observed on changing from potassium to rubidium solution on one side of the red cell membrane white keeping the other side in high potassium. The current-voltage curve in symmetrical potassium solutions exhibited strong inward rectification. When Rb+ was substituted for K+, the I-V curve became almost linear. These findings can all be described using a model with two binding sites, in which the ions are assumed to be both permeating and blocking.

Calcium↗
PubMed1989

Experimental aspects of isolation of myocytes from adult guinea-pig hearts.

Ionic channels can now be effectively studied on enzymatically isolated cardiac myocytes by means of the patch clamp technique. Three procedures reported to give consistently high yields of Ca-tolerant myocytes were tested for applicability to calcium channel studies under our laboratory conditions. None of them was found to be suitable for direct use. Therefore, a modified method for isolation of myocytes from adult guinea-pig hearts was developed. Calcium channel currents measured in Ca-tolerant myocytes isolated by this procedure have been presented and problems of myocytes isolation and of patch-clamp measurement discussed.

Animals↗
PubMed1987

Calcium channels in crayfish muscle fibre fragments studied by means of the Vaseline gap technique.

Calcium currents in crayfish muscle fibres were studied by means of the vaseline gap voltage clamp technique. Overlapping potassium currents were fully suppressed using fibre fragments equilibrated in K+-free intracellular solution. The design of the recording chamber tailored to crayfish muscle fibres is described in detail. Ca currents recorded has a two-component time course. The transient (ICa, T) component (peaking in about 10 ms) attained, on average, maximal overall density of 26.4 microA/cm2 at depolarization to -4.6 mV from a holding potential of -80 mV. The steady (ICa, S) component attained 16.7 microA/cm2 (evaluated at the end of a 70 ms pulse) at +13.8 mV. The average overall surface area of the clamped membrane surface (including invaginated parts) was about 0.07 cm2. The ICa, S component could be separated from ICa, T using short inactivating prepulses. Voltage and time dependence of the transient component inactivation, as well as its recovery from inactivation, were in agreement with a Ca-dependent mechanism. Independent behaviour of the two Ca current components and differences in their properties support the hypothesis concerning the existence of two populations of Ca channels in the surface membrane of the crayfish muscle.

Animals↗