Models of Ca2+ release channel adaptation.
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Adaptation, Physiological↗
Biomedical subjects
F Sachs
Publications and source records attributed to F Sachs.
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↗
Mechanically sensitive, nonselective cation channels.
Mechanically sensitive channels (MSCs) are ubiquitous in plant and animal cells. They respond primarily to membrane tension, thus making them good transducers for forces derived from osmotic or hydraulic gradients and shear stress. They may also be modulated by membrane voltage and various ligands. MSCs are most commonly cation selective, passing calcium as well as monovalent ions, but some are K+ selective, and a few are anion selective. MSCs occur at a density of about 0.2-5 per microns2. The universal distribution and biophysical properties of MSCs make them the ideal mechanotransducers in a wide variety of cellular processes.
Animals↗
Gated, ion-selective channels observed with patch pipettes in the absence of membranes: novel properties of a gigaseal.
Gigaohm seals made between patch pipettes and hydrophobic substrates have a finite conductance which are cation-selective and capable of producing quantized gating indistinguishable from the gating of biological ion channels. The selectivity sequence and streaming potentials of these seals suggests the existence of a pore of similar dimensions to the nicotinic acetylcholine channel. The ionic selectivity of these seals appears similar to the seal selectivity observed with membrane patches (Fischmeister, R., R. K. Ayer, and R. L. DeHann. 1986. Pfluegers Arch. 406:73-82) and the possibility of discrete gating within the seal region suggests caution when interpreting patch clamp data from unfamiliar preparations. The data suggests that the permeation pathway is the narrow space between the hydrophobic substrate and the pipette. Since this space has one hydrophobic wall, a hydrophilic channel lining may not be essential for channel permeation and gating.
Biophysical Phenomena↗
Stretch-activated ion channels in tissue-cultured chick heart.
With use of single-channel patch-clamp recording, we found five distinct types of stretch-activated ion channels (SACs) in tissue-cultured embryonic chick cardiac myocytes. With 140 mM K+ saline in the pipette, four channels had linear conductances of approximately equal to 25, 50, 100, and 200 pS and other channel was an inward rectifier of approximately equal to 25 pS at 0 mV membrane potential. The 100- and 200-pS channels were K+ selective, whereas the others passed alkali cations and Ca2+. From reversal potentials, the permeability ratio of K+/Na+, PK/PNa, was 3-7 for nonselective channels and 7-16 for K(+)-selective channels. Channel density was approximately equal to 0.3/microns2 for linear conductances and approximately equal to 0.1/microns2 for inward rectifier. Open-channel noise was a function of pipette filling solution with root-mean-square (RMS) noise increasing in the order K+ < isosmotic sucrose (plus trace ions) < Na+, probably reflecting short-lived block by extracellular ions. All channels were blocked by 20 microM Gd3+. The 25-pS linear channel was also blocked by 12.5 microM tetrodotoxin and 10 microM diltiazem, but the others were insensitive at these concentrations. Extracellular Cs+ and tetraethylammonium chloride did not block any channels. We saw no SAC activity in cells grown without embryo extract (EE), which demonstrates that channel expression, or some necessary cofactor, is under control of growth factors. Basic fibroblast growth factor (FGF) could replace EE in supporting channel expression. The presence of SACs capable of generating inward currents might explain how stretch increases automaticity in the heart. Because some SACs were permeable to Ca2+, they could contribute to the Starling curve and perhaps to initiating stretch-induced hypertrophy.
Animals↗
Mechanotransducing ion channels in astrocytes.
Ion channels present on the soma of neonatal rat astrocytes in primary cell culture were studied using the single channel recording technique. Ion channels were activated by changing the pressure in the back of the pipette. The morphological structure of the patch membrane was examined while recording channel activity. One class of channel was activated by increasing the pipette pressure (curvature-sensitive or CS channels). CS channels were observed in 150 mM KCl, 150 mM NaCl, or 150 mM sodium gluconate. At constant pressure the closed times decreased with depolarization. CS channels had a conductance of 50 pS in 150 mM NaCl, and displayed an inwardly rectifying current-voltage relationship. CS channel activity was found only in cell-attached patches, and were active only when the patch membrane curved towards the soma. The other class of channel was found to be activated by both suction and pressure (stretch-activated or SA channels). Four SA conductance levels were found: 360, 230, 144, and 70 pS in 150 mM KCl. Each conductance displayed a linear current-voltage relationship. At negative membrane potentials SA channels were inhibited by Cs+, Ba2+ or Na+. The relationship between average mechanosensory current and pressure was biphasic for SA channels and monophasic for CS channels. Combinations of SA and CS channels could be observed in the same patch. We propose that CS channels are non-specific cation channels which sense membrane tension only when the patch membrane is in a specific, permissive curvature. SA channels appear to be K(+)-selective channels that sense membrane tension independent of the direction of curvature.
Animals↗
Calcium imaging of mechanically induced fluxes in tissue-cultured chick heart: role of stretch-activated ion channels.
Heart rate and contractility are sensitive to stretch. To better understand the origin of these effects, we have studied the effect of mechanical stimuli on a model system of tissue-cultured heart cells. Gently prodding cells with a pipette produced a Ca2+ influx that often led to waves of calcium-induced calcium release (CICR) spreading from the site of stimulation. Ca2+ release could also be produced by pulling on neighboring cells. The response was blocked by removing extracellular Ca2+ or by adding 20 microM Gd3+ to normal saline. The mechanical sensitivity probably arose from stretch-activated ion channels (SACs) based on several lines of evidence. Chick heart cells contain nonselective cation SACs that pass Ca2+ as well as Na+ and K+. Both the SACs and the fluorescence response are blocked by 20 microM Gd3+. Removal of Ca2+ from the extracellular medium blocked the fluorescent response. Cultures without SACs (grown in the absence of embryo extract) had no mechanically induced fluxes. These data contradict the recent claim that SAC activity is a patch-clamp artifact (C.E. Morris and R. Horn, Science Wash. DC 256: 1246-1249, 1991). The SACs had a density of approximately 1/micron 2 and were expected to pass less than 20 fA of Ca2+ current under physiological conditions. The change in intracellular concentration of Ca2+ ([Ca2+]i) resulting from activation of SACs may be too small to induce CICR unless the channels pass current into a restricted space (N. LeBlanc and J.R. Hume, Science Wash. DC 248: 372, 1990).(ABSTRACT TRUNCATED AT 250 WORDS)
Aniline Compounds↗
Stretch-sensitive ion channels: an update.
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Animals↗
Inhibiting synthesis of extracellular matrix improves patch clamp seal formation.
The ability to form gigaohm seals is essential for patch-clamp studies. Cells with otherwise useful properties must be abandoned for electrophysiological studies if seal formation is not possible. We have found that by inhibiting the growth of extracellular matrix with beta-D-xyloside, the success of forming gigaohm seals increased from near 0% to near 100%. Treated cells remained viable and appeared morphologically similar to untreated cells. Prototype treatment protocols are given for the renin secreting cell line As4.1.
Animals↗
Alignment of tomographic projections using an incomplete set of fiducial markers.
Reconstruction of three-dimensional images using tomography requires that the projections be aligned along a common rotational axis. We present here a solution to the problem of alignment for single-axis tomography using fiducial markers. The algorithm is based on iterative linearization of the projection equations and is least-squares-optimized by a linear least-squares solution instead of a gradient search. The algorithm does not require markers to be available in every projection, and initial estimates are unnecessary. Program execution is robust, fast, and can quickly align large data sets containing 256 or more projections.
Algorithms↗
Stretch-activated ion channels in guinea pig outer hair cells.
Two types of stretch-activated (SA) ion channels have been found in the lateral wall of isolated outer hair cells (OHC) from the guinea pig cochlea. One type had a reversal potential of -12 mV and was non-selective to cations, passing Ca2+ as well as monovalent ions. The channel had a conductance of 38-50 pS and the amplitude of the current through the open SA channel was independent of suction. The probability of the channel being open increased with applied suction and was voltage dependent with the maximum probability occurring at pipette potentials of -40 to -60 mV. The second type of SA channel had a conductance of approximately 150 pS and a reversal potential of approximately -50 mV. The ionic selectivity of this channel has not yet been determined, but it is probably K+ selective. OHCs have been shown to undergo a slow change in length in response to acoustic stimulation directed at the lateral wall of the OHC. The SA channels reported here could affect the motile response by altering the membrane potential or by allowing the entry of free Ca2+ which could lead to a change in OHC length through the interaction of actin and myosin. SA channels could also play an important role in regulating the osmotic pressure of OHC thereby influencing its electro-mechanical response.
Acoustic Stimulation↗
Quantitative video microscopy of patch clamped membranes stress, strain, capacitance, and stretch channel activation.
Membrane patches from chick skeletal muscle were stretched by applying controlled suction or pressure to the pipette. From images of the patch, the patch dimensions (area and radius of curvature) were computed by nonlinear regression of the images to a geometric model. With no applied pressure, patch membranes are nearly planar and normal to the wall of the pipette. With increasing pressure gradients, the patch bulges, the radius of curvature decreases, and the area increases. The patch capacitance changes in exact proportion to the change in area at a rate of 0.7 microF/cm2. The increase in area is due to a flow of lipid (with perhaps small amounts of diffusible protein) along the walls of the pipette into the patch. The flow is reversible with a relaxation of the pressure gradient. The area elastic constant of the membrane is approximately 50 dyn/cm, insensitive to cytochalasin B and probably represents the elasticity of the underlying spectrin/dystrophin network. Simultaneous measurements of stretch activated (SA) ion channel activity in the patch showed that the sensitivity of channels from different patches, although different when calculated as a function of applied pressure, was the same when calculated as a function of tension. Because patch lipid is free to flow, and hence stress-free in the steady state, SA channels must be activated by tension in the cytoskeleton.
Animals↗
Stochastic models for mechanical transduction.
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Ion Channels↗
The ultrastructure of patch-clamped membranes: a study using high voltage electron microscopy.
We have developed techniques for studying patch-clamped membranes inside glass pipettes using high voltage electron microscopy (HVEM). To preserve the patch structure with the least possible distortion, we rapidly froze and freeze dried the pipette tip. The pipette is transparent for more than 50 microns from the tip. HVEM images of patches confirm light microscopy observations that the patch is not a bare bilayer, but a membrane-covered bleb of cytoplasm that may include organelles and cytoskeleton. The membrane that spans the pipette is commonly tens of micrometers from the tip of the pipette and occasionally as far as 100 microns. The structure of patches taken from a single cell type is variable but there are consistent differences between patches made from different cell types. With suction applied to the pipette before seal formation, we have seen in the light microscope vesicles swept from the plasmalemma up the pipette. These vesicles are visible in electron micrographs, particularly those made from chick cardiac muscle. Colloidal gold labeling of the patch permitted identification of lectin-binding sites and acetylcholine receptors. In young cultures of Xenopus myocytes, the receptors were diffuse. In 1-wk-old cultures, the receptors formed densely packed arrays. The patch pipette can serve, not only as a recording device, but as a tool for sampling discrete regions of the cell surface. Because the pipette has a constant path length for axial rotation, it is a unique specimen holder for microtomography. We have made preliminary tomographic reconstructions of a patch from Xenopus oocyte.
Animals↗
Stretch-activated channels in heart cells: relevance to cardiac hypertrophy.
Stretch-activated channels have been proposed as the transduction mechanism between load and protein synthesis in cardiac hypertrophy. Under this hypothesis, cardiac deformation is linked to an increased sodium (Na) influx, which, in turn, increases protein synthesis. We have tested whether stretch actually increases Na influx by applying patch-clamp techniques to cultured chick embryo cardiac myocytes and to freshly isolated adult guinea pig cardiomyocytes. Our experiments, in excised and cell-attached patches, revealed the existence of ionic channels that opened, or increased their frequency of opening, upon the application of negative pressures to the lumen of the patch-clamp pipettes. These stretch-sensitive channels allowed the passage of the major monovalent physiological cations, Na and potassium (K), and, to a much lesser extent, the major divalent cations calcium (Ca) and magnesium (Mg). Under normal conditions, the channels had a high open channel noise that prevented the customary, straightforward statistical analysis of single channel data. However, when one of the major monovalent cations was iso-osmotically replaced by sucrose, the open channel noise decreased significantly and permitted a good delineation of the open and closed channel states and, therefore, application of standard patch-clamp, statistical analysis techniques. Under these "sucrose," "monoionic" conditions, the reversal potential was, as one should expect, close to the equilibrium potential for the major monovalent cation present. When high extracellular K solution was used to minimize the cell resting potential, the reversal potential for these stretch-activated currents was estimated to be around -40 mV. Therefore, under normal conditions, stretch should induce an inward, depolarizing current, carried mostly by Na ions.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Stretch-activated ion channels and membrane mechanics.
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Animals↗
The structure and dynamics of patch-clamped membranes: a study using differential interference contrast light microscopy.
We have developed techniques for micromanipulation under high power video microscopy. We have used these to study the structure and motion of patch-clamped membranes when driven by pressure steps. Patch-clamped membranes do not consist of just a membrane, but rather a plug of membrane-covered cytoplasm. There are organelles and vesicles within the cytoplasm in the pipette tip of both cell-attached and excised patches. The cytoplasm is capable of active contraction normal to the plane of the membrane. With suction applied before seal formation, vesicles may be swept from the cell surface by shear stress generated from the flow of saline over the cell surface. In this case, patch recordings are made from membrane that was not originally present under the tip. The vesicles may break, or fuse and break, to form the gigasealed patch. Patch membranes adhere strongly to the wall of the pipette so that at zero transmural pressure the membranes tend to be normal to the wall. With transmural pressure gradients, the membranes generally become spherical; the radius of curvature decreasing with increasing pressure. Some patches have nonuniform curvature demonstrating that forces normal to the membrane may be significant. Membranes often do not respond quickly to changes in pipette pressure, probably because viscoelastic cytoplasm reduces the rate of flow through the tip of the pipette. Inside-out patches may be peeled from the walls of the pipette, and even everted (with positive pressure), without losing the seal. This suggests that the gigaseal is a distributed property of the membrane-glass interface.
Animals↗
Characterization of stretch-activated ion channels in Xenopus oocytes.
1. The gating and permeation properties of endogenous stretch-activated (SA) ion channels in Xenopus oocytes have been studied using the patch-clamp single channel recording technique. 2. As estimated from the probability of being open (Po), SA channels were equally sensitive to suction or pressure. The Po was also weakly sensitive to voltage, increasing with depolarization. Channel activation did not require Ca2+. 3. Kinetic analysis of single-channel records indicated that there are three closed states and one open state. Among three closed-time distributions, the longest was the most sensitive to both pipette pressure and membrane voltage. The open time was independent of both pressure and voltage under a wide variety of ionic conditions, but was sensitive to the species of extracellular ion as follows: Na+ greater than Cs+ greater than K+ greater than Rb+ greater than Li+. The open time had a monotonic mole fraction relationship in mixtures of Li+ and K+. 4. The SA channels were cation-selective inward rectifiers. The selectivity for permeation, based on slope conductance, was: K+ greater than NH4+ greater than Cs+ greater than Rb+ greater than Na+ greater than Li+ greater than Ca2+. 5. Tetraethylammonium (TEA+) was impermeable but was not a channel blocker. 6.Open-channel current amplitude saturated with increasing extracellular K+, and was a monotonic function of the mole fraction of Li+ and K+ in mixtures of the two ions. 7. The channel has at least two separate ion binding sites: an intra-channel site suggested by the permeation data, and an allosteric site suggested by the voltage-independent effects of permeant ions on open time. A symmetric two-barrier, one-site model can quantitatively describe the permeation data. A kinetic model is proposed to quantify the gating kinetics and the effect of ion binding at the allosteric site.
Action Potentials↗