Identification of ion channel regulating proteins by patch-clamp analysis.
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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↗
Comparisons of somatic action potentials from dispersed and intact rat nodose ganglia using patch-clamp technique.
AIM: To differentiate the electrophysiological characteristics of somatic action potentials (AP) from isolated Neo and Juv nodose sensory neurons (NSN) and those from slices of intact Juv and adult rat nodose ganglia. METHODS: For isolated cell recordings nodose ganglia from 3-8 d old Neo and 4 weeks old Juv rats were dissociated using trypsin and collagenase, respectively. Nodose ganglia slices with attached vagus were prepared using a sequential treatment of collagenase and trypsin for both Juv and adult rats. Conduction velocity (CV) was collected by vagal stimulation. Whole-cell patch was applied for somatic AP recordings. RESULTS: (1) 281 NSN from both isolated cells and nodose slices were studied. Across all age groups, there was no difference observed among either C- or A-types. The difference between C- and A-type was significant. (2) Neurons exhibiting AP with prominent repolarization hump, broader APD50 (>2.0 ms), upstroke velocity at the point of APD50 (UV(APD50)) and downstroke velocity at the point of APD50 (DV(APD50)) below 100 V . s-1 and 50 V . s-1 were classified as C-type (n = 222). Those without a hump, brief APD50 (<1.0 ms), and UV(APD50) and DV(APD50) greater than 200 V . s-1 and 100 V . s-1 were classified as A-type (n = 59). (3) With slices, except for hump, APD, UV, and DV, significant differences were also observed (C- vs A-type) in CV from both Juv (0.56 +/- 0.15 vs 15.6 m.s-1) and adult (0.9 +/- 0.4 vs 14.5 +/- 1.0 m . s-1) nodose slices, discharge rate (single or burst vs repetitive), and frequency follow (<20 Hz vs >100 Hz). (4) Phase analysis showed that C-type had higher AP firing threshold, and lower total in- and outward peak currents than A-type. CONCLUSION: C- and A-type AP from isolated NSN of Neo or Juv rats exhibited electrophysiological characteristics that closely followed those of AP recorded in nodose slices. Therefore, isolated NSN can effectively serve as an experimental surrogate for electrophysiological studies of NSN requiring prior identification of afferent fiber type. Features of the AP wave-form such as the repolarization hump, APD50), and UV are strongly correlated with CV and are therefore reliable measures for classifying isolated NSN as either C- or A-type. It is most important that the nodose slice enables us to study the AP and ion channel characteristics on identified NSN by afferent fiber CV with adult animals.
Action Potentials↗
Mechanism of heptanol-induced uncoupling of cardiac gap junctions: a perforated patch-clamp study.
The influence of heptanol on gap junctional and non-junctional membrane currents was studied in cultured neonatal rat heart cells using both the whole cell and perforated patch voltage-clamp method. With both methods, exposure to heptanol produced a dose-dependent decrease in the junctional current (dissociation constant = 0.54 and 1.20 mM for whole cell and perforated patch experiments, respectively). Heptanol-induced uncoupling was reversible. In the same concentration range, heptanol reduced all nonjunctional membrane ionic currents examined. This suggests that heptanol does not act specifically on gap junction channels but rather on the structure of the lipid membrane. This hypothesis is strengthened by the observation that in monolayer cultures of neonatal rat heart cells fluorescence steady-state anisotropy decreased proportional with increasing the heptanol concentration in the bath. Single-channel conductances (gamma j) were identical with both recording methods (21 and 40-45 pS); heptanol did not alter gamma j. Under conditions of reduced junctional coupling induced by heptanol, junctional conductance (gj) displayed voltage sensitivity at values of gj at which no voltage sensitivity could be observed under control conditions. These results suggest that heptanol-dependent uncoupling was caused by a decrease in open probability of the gap junction channels.
Alcohols↗
Patch-clamp studies of cystic fibrosis transmembrane conductance regulator chloride channel.
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Adenosine Triphosphate↗
[A two-compartment model for the dependence of a postsynaptic potential on a postsynaptic current, measured by the patch-clamp method].
As known, the dependence of a postsynaptic potential (PSP)1 on a postsynaptic current (PSC) is not satisfactorily approximated by simple Ohm's law due to a significant role of electrotonic propagation of currents along dendrites. The present work shows that a two-compartment model of a neuron, conjointly solving the two problems of voltage and current clamping, gives quite precisely the PSP-on-PSC dependence, in spite of inaccurate reconstruction of currents on dendritic terminals. The two-compartment model is compared with the neuron model consisting of a distributed cylindrical dendrite and a concentrated soma.
Animals↗
A system for applying rapid warming or cooling stimuli to cells during patch clamp recording or ion imaging.
We describe a system for superfusing small groups of cells at a precisely controlled and rapidly adjustable local temperature. Before being applied to the cell or cells under study, solutions are heated or cooled in a chamber of small volume ( approximately 150 microl) and large surface area, sandwiched between four small Peltier elements. The current through the Peltier elements is controlled by a microprocessor using a PID (proportional-integral-derivative) feedback algorithm. The chamber can be heated to at least 60 degrees C and cooled to 0 degrees C, changing its temperature at a maximum rate of about 7 degrees C per second; temperature ramps can be followed under feedback control at up to 4 degrees C per second. Temperature commands can be applied from the digital-to-analogue converter of any laboratory interface or generated digitally by the microprocessor. The peak-to-peak noise contributed by the system does not exceed that contributed by a patch pipette, holder and headstage, making it suitable for single channel as well as whole cell recordings.
Action Potentials↗
Whole cell patch-clamp study of putative vasomotor neurons isolated from the rostral ventrolateral medulla.
A distinct subpopulation of neurons in the rostral and ventrolateral part of the medulla oblongata (RVL) plays a key role in controlling sympathetic vasomotor tone. To characterize these neurons under conditions in which all cell-to-cell interactions are eliminated, RVL neurons were acutely dissociated from 13- to 19-day old rats. Cells projecting to the upper thoracic segments were retrogradely labeled with fluorescent beads. Fifty-two percent (17/33) of examined spinally projecting neurons were catecholaminergic, as demonstrated by single-cell reverse transcription-polymerase chain reaction or immunocytochemistry. No spontaneous (capacitive) spikes were revealed in the tight seal cell-attached configuration. Whole cell recordings were made from 54 spinally projecting neurons using Cs(+)- or K(+)-containing pipettes. No spontaneous firing was observed in current-clamp mode with K(+)-based pipettes (membrane potential, -61.5 +/- 2.3 mV). Step depolarizations (300- or 400-ms pulses, up to 100 pA) evoked regular firing or one to four spikes. Several voltage-gated currents, resembling the transient and persistent Na+, delayed rectifier and low- and high-threshold Ca2+, were revealed in voltage-clamp mode. These results show that isolated spinally projecting RVL neurons display no pacemaker-like activity. Because data from the literature indicate that these neurons are capable of generating such activity under different experimental conditions, the factors responsible for different behavior need to be determined. Dissociated RVL neurons provide a useful new model for studying biophysical and other properties of neurons involved in blood pressure control.
Animals↗
Direct measurement of the voltage sensitivity of second-harmonic generation from a membrane dye in patch-clamped cells.
We report what is to our knowledge the first optical imaging of voltage-clamped cells by second-harmonic generation. For the membrane-staining styryl dye di-4-ANEPPS, we determined the sensitivity of second-harmonic generation to be 18%/100 mV at an excitation wavelength of 850 ns. This sensitivity is significantly better than the optimal 10%/100 mV under fluorescence and further establishes the importance of second-harmonic generation for the functional imaging of membrane potential in living cells.
Animals↗
A patch-clamp investigation of membrane currents in a novel mammalian retinal ganglion cell line.
We characterised membrane currents in undifferentiated RGC-5 cells, a cell line used in in vitro models of apoptosis and glaucoma. The cells were inexcitable, with no voltage-dependent Na(+) currents or action potentials. Some novel currents were observed including basal Cl(-) currents, inwardly rectifiying K(+) currents and Gd(3+) insensitive stretch-activated currents. Our results highlight the differences between the electrophysiological properties of undifferentiated RGC-5 cells and retinal ganglion cells.
Animals↗
Patch clamped single pancreatic zymogen granules: direct measurements of ion channel activities at the granule membrane.
BACKGROUND/AIM: Pancreatic acinar cells are involved in the secretion of digestive enzymes. Digestive enzymes in pancreatic acinar cells are stored in membrane-bound secretory vesicles called zymogen granules (ZGs). The swelling of ZGs is implicated in the regulation of the expulsion of intravesicular contents during secretion. The molecular mechanism of ZG swelling has been previously elucidated. It has been further demonstrated that the water channel aquaporin-1, the potassium channel IRK-8, and the chloride channel CLC-2, are present in the ZG membrane and involved in ZG swelling. However, a direct measurement of these ion channels at the ZG membrane in intact ZGs had not been performed. The aim of this study was to investigate the electrical activity of single ZGs and verify the types of channels found within their membrane. METHODS: ZGs from pancreatic acinar cells were isolated from the pancreas of Sprague-Dawley rats. Direct measurements of whole vesicle currents, in the presence and absence of ion channel blockers (quinine, glyburide and DIDS), were recorded following successful patching of single ZGs. CONCLUSION: In this study, we were able, for the first time, to patch single ZGs and study ion channels in their membrane. We were able to record currents across the ZG membrane and, utilizing ion channel blockers, confirm the presence of the chloride channels CLC-2 and the potassium channel IRK-8 (Kir6.1), and additionally demonstrate the presence of a second chloride channel CLC-3.
ATP-Binding Cassette Transporters↗
Patch-clamp studies in the CNS illustrate a simple new method for obtaining viable neurons in rat brain slices: glycerol replacement of NaCl protects CNS neurons.
Viable neurons in brain slices are crucial for electrophysiological studies. The present study describes a new method for obtaining viable cells in several regions of the central nervous system including the ventral tegmental area, the hypothalamus, the periaqueductal grey matter and the spinal cord. The essence of the method was to use a modified artificial cerebrospinal fluid (ACSF) in which all NaCl was replaced initially by equi-osmotic glycerol. This modified glycerol-based ACSF was used during slice preparation. The underlying principle for the modification is to prevent the possible acute neurotoxic effects of passive chloride entry, subsequent cell swelling and lysis. This method significantly increased the live/dead ratio in morphology compared to the normal ACSF or sucrose-base ACSF, in which NaCl was replaced by sucrose. An examination of some electrophysiological and pharmacological properties of the neurons in these preparations, by means of current-clamp and voltage-clamp recordings, revealed similar properties of those neurons obtained with the traditional ACSF method. Due to the increase in the number of viable neurons, the new ACSF increases the productivity of experiments. Based on our data, we propose that this glycerol-based solution may protect CNS neurons.
Animals↗
Patch-clamp investigations and compartmental modeling of rod bipolar axon terminals in an in vitro thin-slice preparation of the mammalian retina.
To extend the usefulness of rod bipolar cells for studies of chemical synaptic transmission, we have performed electrophysiological recordings from rod bipolar axon terminals in an in vitro slice preparation of the rat retina. Whole cell recordings from axon terminals and cell bodies were used to investigate the passive membrane properties of rod bipolar cells and analyzed with a two-compartment equivalent electrical circuit model developed by Mennerick et al. For both terminal- and soma-end recordings, capacitive current decays were well fitted by biexponential functions. Computer simulations of simplified models of rod bipolar cells demonstrated that estimates of the capacitance of the axon terminal compartment can depend critically on the recording location, with terminal-end recordings giving the best estimates. Computer simulations and whole cell recordings demonstrated that terminal-end recordings can yield more accurate estimates of the peak amplitude and kinetic properties of postsynaptic currents generated at the axon terminals due to increased electrotonic filtering of these currents when recorded at the soma. Finally, we present whole cell and outside-out patch recordings from axon terminals with responses evoked by GABA and glycine, spontaneous inhibitory postsynaptic currents, voltage-gated Ca(2+) currents, and depolarization-evoked reciprocal synaptic responses, verifying that the recorded axon terminals are involved in normal pre- and postsynaptic relationships. These results demonstrate that axon terminals of rod bipolar cells are directly accessible to whole cell and outside-out patch recordings, extending the usefulness of this preparation for detailed studies of pre- and postsynaptic mechanisms of synaptic transmission in the CNS.
Animals↗
Dependence of solution exchange time on cell or patch linear dimensions in concentration jump experiments using patch-clamped sensory neurones.
A critical evaluation of "concentration jump" techniques employing hydrodynamic solution exchange has been undertaken. Data obtained from experiments using acutely isolated sensory neurones demonstrated a dependence of the total solution exchange time on the linear dimensions of the preparation. The exchange time around a circular surface is proportional to the radius of this surface. A quantitative approximation can be formulated as follows: if the membrane area of the object under investigation is increased n times, the duration of the full solution exchange is increased by radical n times. A nonlinear dependence of exchange time on vacuum pressure was confirmed in a separate set of experiments.
Animals↗
[Research of the electrophysiological properties of acutely dissociated spiral ganglion neurons isolated from mice using patch clamp technique].
OBJECTIVE: To explore the electrophysiological properties of spiral ganglion neurons (SGN) isolated from neonatal mice. METHODS: Ion channel currents of acutely trypsin-dissociated spiral ganglion neurons was recorded and analyzed using whole-cell variation of the voltage clamp technique. RESULTS: In the membrane of SGN, we recorded tetraethylammonium (TEA)-sensitive and 4-aminopyridine (4-AP)-sensitive outside delayed rectifier potassium currents, A-type potassium currents and tetrodotoxin-sensitive inside sodium currents. Ion channel activity had the character of voltage-dependence. Some cells, which did not exhibit sodium currents exhibited, delayed rectifier potassium currents. CONCLUSIONS: The electrophysiological properties of acutely dissociated spiral ganglion neurons could be reference to the research of the mechanics of auditory propagation and the ion channel pharmacology.
Animals↗
Confocal microscopic detection of potential-sensitive dyes used to reveal loss of voltage control during patch-clamp experiments.
We used a fast, fluorescent, potential-sensitive indicator (Di-8-ANEPPS) in combination with laser-scanning confocal microscopy in the line-scan mode (temporal resolution 500 Hz) to independently determine the transmembrane potential in voltage-clamped cells. While a linear relation between command voltage and Di-8-ANEPPS fluorescence was found in unexcitable Sf9 cells, pronounced nonlinearities were observed in cardiac myocytes. Comparison of the fluorescence records and current traces indicated that most of the observed nonlinearities could be attributed to voltage-escape during flow of membrane current. Voltage-escape during large membrane currents may lead to various experimental difficulties during voltage-clamp experiments. The voltage recording technique based on fluorescence was then used to compare the voltage-escape during flow of Na+ and Li+ ions via voltage-dependent (TTX sensitive) Na+ channels in cardiac myocytes. In these experiments, no significant differences in the degree of voltage-escape was found, suggesting that the two currents were similar in amplitude. In addition to the application presented in this paper, confocal microscopic detection of transmembrane potential with fluorescent dyes may be a useful technique for experiments in preparations that are difficult to impale with microelectrodes because of their small size.
Animals↗
Silicon chip-based patch-clamp electrodes integrated with PDMS microfluidics.
We report on a silicon wafer-based device that can be used for recording macroscopic ion channel protein activities across a diverse group of cell-types. Gigaohm seals were achieved for CHO-K1 and RIN m5F cells, and both cell-attached and whole-cell mode configurations were also demonstrated. Two distinct intrinsic potassium ion channels were recorded in whole-cell mode for HIT-T15 and RAW 264.7 cells. Polydimethylsiloxane (PDMS) microfluidics were also coupled with the micromachined silicon chips in order to demonstrate that a single cell could be selectively directed to a micropore, and membrane protein currents could subsequently be recorded. These silicon chip-based devices have significant advantages over traditional micropipette approaches, and may serve as combinatorial tools for investigating membrane biophysics, pharmaceutical screening, and other bio-sensing tasks.
Animals↗
Optimization of a multi-channel puffer system for rapid delivery of solutions during patch-clamp experiments.
In biological experiments, especially in neuroscience research, it is important to manipulate the extracellular environment efficiently. We have developed a micro-puffing system for local drug delivery to single cells in electrophysiological experiments, and validated the kinetic properties of this instrument. Based on our results, the kinetics of the delivery of solutions and the territory controlled by this system are influenced by several factors: (1) inner diameter (I.D.) of the guide tubing; (2) I.D. of the puffing tip; (3) angle of the puffing tip; and (4) gravity or external pressure applied to the solution. The system can fully control a territory of 200 x 600 micrometer2. The minimum delay in response to drug delivery is 10-20 ms. Switching between different solutions takes less than 100 ms. The minimum volume of solution required by the system is 0.2 ml. Taken together, our results provide useful data for designing and using an efficient drug/solution delivery system in electrophysiological experiments.
Animals↗