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Biomedical subjects

F Sachs

Publications and source records attributed to F Sachs.

At least 37 records · Page 2Linked to original sources
PubMed1999

Single channel properties of P2X2 purinoceptors.

The single channel properties of cloned P2X2 purinoceptors expressed in human embryonic kidney (HEK) 293 cells and Xenopus oocytes were studied in outside-out patches. The mean single channel current-voltage relationship exhibited inward rectification in symmetric solutions with a chord conductance of approximately 30 pS at -100 mV in 145 mM NaCl. The channel open state exhibited fast flickering with significant power beyond 10 kHz. Conformational changes, not ionic blockade, appeared responsible for the flickering. The equilibrium constant of Na+ binding in the pore was approximately 150 mM at 0 mV and voltage dependent. The binding site appeared to be approximately 0.2 of the electrical distance from the extracellular surface. The mean channel current and the excess noise had the selectivity: K+ > Rb+ > Cs+ > Na+ > Li+. ATP increased the probability of being open (Po) to a maximum of 0.6 with an EC50 of 11.2 microM and a Hill coefficient of 2.3. Lowering extracellular pH enhanced the apparent affinity of the channel for ATP with a pKa of approximately 7.9, but did not cause a proton block of the open channel. High pH slowed the rise time to steps of ATP without affecting the fall time. The mean single channel amplitude was independent of pH, but the excess noise increased with decreasing pH. Kinetic analysis showed that ATP shortened the mean closed time but did not affect the mean open time. Maximum likelihood kinetic fitting of idealized single channel currents at different ATP concentrations produced a model with four sequential closed states (three binding steps) branching to two open states that converged on a final closed state. The ATP association rates increased with the sequential binding of ATP showing that the binding sites are not independent, but positively cooperative. Partially liganded channels do not appear to open. The predicted Po vs. ATP concentration closely matches the single channel current dose-response curve.

Adenosine Triphosphate↗
PubMed1998

The breakdown of cell membranes by electrical and mechanical stress.

We attempted to determine whether mechanical tension and electrical stress couple to cause membrane breakdown in cells. Using cell-attached patches from HEK293 cells, we estimated the mechanically produced tension from the applied pressure and geometry of the patch. Voltage pulses of increasing amplitude were applied until we observed a sudden increase in conductance and capacitance. For pulses of 50 micros duration, breakdown required >0.5 V and was dependent on the tension. For pulses of 50-100 ms duration, breakdown required 0.2-0.4 V and was independent of tension. Apparently two physically different processes can lead to membrane breakdown. We could explain the response to the short, high-voltage pulses if breakdown occurred in the lipid bilayer. The critical electromechanical energy per unit area for breakdown by short pulses was approximately 4 dyne/cm, in agreement with earlier results on bilayers. Our data suggest that, at least in a patch, the bilayer may hold a significant fraction (approximately 40%) of the mean tension. To be compatible with the large, nonlytic area changes of patches, the bilayer appears to be pulled toward the pipette tip, perhaps by hydrophobic forces wetting membrane proteins bound to the glass. Although breakdown voltages for long pulses were in agreement with earlier work on algae, the mechanism(s) for this breakdown remain unclear.

Biophysical Phenomena↗
PubMed1998

Voltage-dependent membrane displacements measured by atomic force microscopy.

Cells use polar molecules in the membrane to sense changes in the transmembrane potential. The opening of voltage-gated ion channels and membrane bending due to the inverse flexoelectric effect are two examples of such electromechanical coupling. We have looked for membrane motions in an electric field using atomic (or scanning) force microscopy (AFM) with the intent of studying voltage-dependent conformational changes of ion channels. Voltage-clamped HEK293 cells were either untransfected controls or transfected with Shaker K+ channels. Using a +/- 10-mV peak-peak AC carrier stimulus, untransfected cells moved 0.5-15 nm normal to the plane of the membrane. These movements tracked the voltage at frequencies >1 kHz with a phase lead of 60-120 degrees, as expected of a displacement current. The movement was outward with depolarization, but the holding potential only weakly influenced the amplitude of the movement. In contrast, cells transfected with a noninactivating mutant of Shaker K+channels showed similar movements, but these were sensitive to the holding potential; decreasing with depolarization between -80 and 0 mV. Searching for artifactual origins of these movements, we used open or sealed pipettes and AFM cantilever placements just above the cells. These results were negative, suggesting that the observed movements were produced by the cell membrane rather than by movement of the patch pipette, or by acoustic or electrical interactions of the membrane with the AFM tip. In control cells, the electrical motor may arise from the flexoelectric effect, where changes in potential induce changes in curvature. In transfected cells, it appears that channel-specific movements also occurred. These experiments demonstrate that the AFM may be able to exploit voltage-dependent movements as a source of contrast for imaging membrane components. The electrically induced motility will cause twitching during action potentials, and may have physiological consequences.

Artifacts↗
PubMed1997

Storing analog data in a video record.

We have designed a simple device that will encode, in machine readable form, multiple analog data in a video record. The analog data is visible to the user within the video frame permitting visual correlation of these signals with activity observed in the image. By superimposition of both analog and video data sets, the two are tightly synchronized and remain so in all copies of the data. The analog data can be separated from the image following digitization by a frame grabber. The bandwidth for each of the five analog channels is approximately 5 kHz. The device, which is essentially an eight channel video multiplexor, includes a video channel, a field counter, an amplitude calibration signal and five analog data channels. The amplitude calibrator allows corrections for gain errors that are particularly prevalent when data is stored on video tape.

Calibration↗
PubMed1997

Maximum likelihood estimation of aggregated Markov processes.

We present a maximum likelihood method for the modelling of aggregated Markov processes. The method utilizes the joint probability density of the observed dwell time sequence as likelihood. A forward-backward recursive procedure is developed for efficient computation of the likelihood function and its derivatives with respect to the model parameters. Based on the calculated forward and backward vectors, analytical formulae for the derivatives of the likelihood function are derived. The method exploits the variable metric optimizer for search of the likelihood space. It converges rapidly and is numerically stable. Numerical examples are given to show the effectiveness of the method.

Markov Chains↗
PubMed1996

Mechanically activated currents in chick heart cells.

As predicted from stretch-induced changes of rate and rhythm in the heart, acutely isolated embryonic chick heart cells exhibit whole-cell mechanosensitive currents. These currents were evoked by pressing on cells with a fire polished micropipette and measured through a perforated patch using a second pipette. The currents were carried by Na+ and K+ but not Cl-, and were independent of external Ca2+. The currents had linear I/V curves reversing at -16 mV and were completely blocked by Gd3+ >/= 30 microM and Grammostola spatulata venom at a dilution of 1:1000. Approximately 20% of cells showed time dependent inactivation. In contrast to direct mechanical stimulation, hypotonic volume stress produced an increase in conductance for anions rather than cations-the two stimuli are not equivalent. The cells had two types of stretch-activated ion channels (SACs): a 21 pS nonspecific cation-selective reversing at -2 mV and a 90 pS K+ selective reversing at -70 mV in normal saline. The activity of SACs was strongly correlated with the presence of whole-cell currents. Both the whole-cell currents and SACs were blocked by Gd3+ and by Grammostola spatulata spider venom. Mechanical stimulation of spontaneously active cells increased the beating rate and this effect was blocked by Gd3+. We conclude that physiologically active mechanosensitive currents arise from stretch activated ion channels.

Action Potentials↗
PubMed1996

Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events.

We present here a maximal likelihood algorithm for estimating single-channel kinetic parameters from idealized patch-clamp data. The algorithm takes into account missed events caused by limited time resolution of the recording system. Assuming a fixed dead time, we derive an explicit expression for the corrected transition rate matrix by generalizing the theory of Roux and Sauve (1985, Biophys. J. 48:149-158) to the case of multiple conductance levels. We use a variable metric optimizer with analytical derivatives for rapidly maximizing the likelihood. The algorithm is applicable to data containing substates and multiple identical or nonidentical channels. It allows multiple data sets obtained under different experimental conditions, e.g., concentration, voltage, and force, to be fit simultaneously. It also permits a variety of constraints on rate constants and provides standard errors for all estimates of model parameters. The algorithm has been tested extensively on a variety of kinetic models with both simulated and experimental data. It is very efficient and robust; rate constants for a multistate model can often be extracted in a processing time of approximately 1 min, largely independent of the starting values.

Algorithms↗
PubMed1996

Ca2+ uptake in GH3 cells during hypotonic swelling: the sensory role of stretch-activated ion channels.

Hypotonic cell swelling triggers an increase in intracellular Ca2+ concentration that is deemed responsible for the subsequent regulated volume decrease in many cells. To understand the mechanisms underlying this increase, we have studied the Ca2+ sources that contribute to hypotonic cell swelling-induced Ca2+ increase (HICI) in GH3 cells. Fura 2 fluorescence of cell populations revealed that extracellular, but not intracellular, stores of Ca2+ were required. HICI was abolished by nifedipine, a blocker of L-type Ca2+ channels, and Gd3+, a nonspecific blocker of stretch-activated channels (SACs), suggesting two components for the Ca2+ membrane pathway: L-type Ca2+ channels and SACs. Using HICI as an assay, we found that venom from the spider Grammostola spatulata could block HICI without blocking L-type Ca2+ channels. The venom did, however, block SAC activity. This suggests that Ca(2+)-permeable SACs, rather than L-type Ca2+ channels, are the sensing elements for HICI. These results support the model for volume regulation in which SACs, activated by an increase of the membrane tension during hypotonic cell swelling, trigger HICI, leading to a volume decrease.

Animals↗
PubMed1996

Stretch-induced voltage changes in the isolated beating heart: importance of the timing of stretch and implications for stretch-activated ion channels.

OBJECTIVES: It is now well recognized that myocardial stretch can cause arrhythmias due to stretch-induced depolarizations. The effects of transient stretch applied during the various phases of the cardiac action potential have not been investigated. This study (1) examined the effects of short stretch pulses and sustained stretch on the monophasic action potential (MPA) repolarization time course and diastolic potential, (2) examined the arrhythmic response to differently timed stretch pulses, and (3) tested by comparison with computer simulations whether these effects are compatible with stretch-activated channel characteristics known from patch-clamp studies. METHODS: We studied the MAP changes elicited by short transient stretch pulses applied at different times during the cardiac cycle to 8 isolated Langendorff-perfused rabbit hearts. The left ventricle (LV) was instrumented with a fluid-filled balloon, the volume of which was altered rapidly and precisely by means of a computer-controlled linear motor-driven piston. MAPs were recorded simultaneously from one right ventricular (RV) and two LV sites while short volume pulses of increasing amplitude were applied to the LV at variable delays after the last of 8 regular electrical pacing stimuli. The effect of pulsatile volume pulses applied at different phases of electrical systole and diastole was compared to the effect of sustained stretch pulses (60 s duration) of the same amplitude. The experimental results were compared with computer simulations of stretch-induced effects on the action potential to further validate the experimentally measured effects with theoretical predictions based on the Oxford Heart model with added stretch channel terms. RESULTS: Stretch pulses applied during early systole caused a brief transient repolarization during the LV MAP plateau phase, with a maximal amplitude of 24 +/- 10% of the total MAP amplitude. Stretch pulses at the end of the MAP caused a transient depolarization, with a maximal amplitude of 13 +/- 5%. These oppositely polarized stretch effects crossed over during a transitional range of repolarization (mean 65 +/- 9% of repolarization) when stretch produced neither transient repolarizations nor depolarizations. Only stretch pulses applied at a mean repolarization level of 77 +/- 5% or later led to arrhythmias, preceded by transient depolarizations. No corresponding de- or repolarizations were seen in MAPs recorded simultaneously from the unstretched RV. The effects of long pulses on the MAP waveform were nearly identical to an overlay plot of the effects of many differently timed short transient pulses. When the stretch-induced voltage changes in the MAP were plotted against the repolarization level at which they were produced, a linear relationship was found (mean correlation coefficient r = 0.97; P < 0.0001) with a reversal at approximately half the total MAP amplitude. The computer simulations of the influence of stretch-activated channels reproduced both the effects of short and sustained stretch seen in the MAP recordings. CONCLUSIONS: We demonstrated in the isolated beating heart that the electrophysiologic effects of sudden myocardial stretch depend on the timing of the stretch relative to electrical systole or diastole. These findings are in agreement with patch clamp studies on stretch-activated ion channels which showed a linear current/voltage relation with a reversal potential between -20 and -30 mV. Only stretch pulses applied at the end of the action potential or during diastole elicit ectopic beats as a result of transient depolarizations, while stretch pulses applied during phase 2 and 3 cause transient repolarizations or no effect, respectively.

Action Potentials↗
PubMed1995

A low drift micropipette holder.

A novel micropipette holder has been designed to overcome the problem of drift encountered when experimenting on small preparations using patch-clamp techniques. In order to increase the stiffness and thermal stability of the system, the holder is designed such that the micropipette is clamped to a rigid quartz rod, and isolated from stresses arising in the connectors. The pressure line, made from stiff tubing, and electrical leads are firmly attached to the table and micromanipulator. This design was able to hold the micropipette tip stable to with 100 nm for more than 1 h, even with changes of 26.7 kPa (200 mmHg) in the pressure line.

Amplifiers, Electronic↗