Internal Patch Pipette Buffer

2014 ◽  
Vol 2014 (6) ◽  
pp. pdb.rec083071-pdb.rec083071
Keyword(s):  
Patch Pipette

The volume-sensitive organic osmolyte-anion channel VSOAC is regulated by nonhydrolytic ATP binding

1994 ◽  
Vol 267 (5) ◽  
pp. C1203-C1209 ◽  
Author(s):  
P. S. Jackson ◽  
R. Morrison ◽  
K. Strange
Keyword(s):  
Ketone Bodies ◽  
Anion Channel ◽  
Metabolic Inhibitors ◽  
Cell Swelling ◽  
Organic Osmolytes ◽  
Pipette Solution ◽  
Organic Osmolyte ◽  
Atp Levels ◽  
Patch Pipette ◽  
Taurine Efflux

Efflux of intracellular organic osmolytes to the external medium is a ubiquitous response to cell swelling. Accumulating evidence indicates that volume regulatory loss of structurally unrelated organic osmolytes from cells is mediated by a relatively nonselective volume-sensitive anion channel. In C6 cells, we have termed this channel VSOAC for volume-sensitive organic osmolyte-anion channel. Swelling-induced activation of VSOAC required the presence of ATP or nonhydrolyzable ATP analogues [adenosine 5'-O-(3-thiotriphosphate), adenylylmethyl-enediphosphonate (AMP-PCP), or 5'-adenylylimidodiphosphate] in the patch pipette. Sustained activation of VSOAC also required ATP. Channel rundown was observed when cellular ATP levels were lowered by intracellular dialysis with the patch pipette solution. Rundown was prevented by the ATP analogue AMP-PCP. Passive swelling-induced myo-[3H]inositol and [3H]taurine efflux was blocked by metabolic inhibitors that decreased cellular ATP levels. Titration of cellular ATP levels with azide demonstrated that the apparent dissociation constant (Kd) for ATP of both myo-inositol and taurine efflux was approximately 1.7 mM. The high Kd for ATP indicates that cellular metabolic state plays an important role in modulating organic osmolyte loss. Regulation of VSOAC activity by ATP prevents depletion of metabolically expensive organic osmolytes when cellular energy production is reduced. In addition, ATP-dependent regulation provides essential feedback to minimize the loss of energy-producing carbon sources such as pyruvate, short-chain fatty acids, ketone bodies, and amino acids, which readily permeate this channel.

scholarly journals Depolarization-Induced Ca2+ Entry Preferentially Evokes Release of Large Quanta in the Developing Xenopus Neuromuscular Junction

2010 ◽  
Vol 104 (5) ◽  
pp. 2730-2740 ◽  
Author(s):  
Xiao-Ping Sun ◽  
Bo-Ming Chen ◽  
Olav Sand ◽  
Yoshi Kidokoro ◽  
Alan D. Grinnell
Keyword(s):  
Synaptic Vesicles ◽  
Channel Blocker ◽  
Direct Injection ◽  
External Solution ◽  
Neuromuscular Synapses ◽  
Quantal Size ◽  
Amplitude Histogram ◽  
Wide Range ◽  
Patch Pipette ◽  
Second Peak

The amplitude histogram of spontaneously occurring miniature synaptic currents (mSCs) is skewed positively at developing Xenopus neuromuscular synapses formed in culture. To test whether the quantal size of nerve-evoked quanta (eSCs) distributes similarly, we compared the amplitude histogram of single quantum eSCs in low external Ca2+ with that of mSCs and found that nerve stimulation preferentially released large quanta. Depolarization of presynaptic terminals by elevating [K+] in the external solution or by direct injection of current through a patch pipette increased the mSC frequency and preferentially, but not exclusively, evoked the release of large quanta, resulting in a second broad peak in the amplitude histogram. Formation of the second peak under these conditions was blocked by the N-type Ca2+ channel blocker, ω-conotoxin GVIA. In contrast, when the mSC frequency was elevated by thapsigargin- or caffeine-induced mobilization of internal Ca2+, formation of the second peak did not occur. We conclude that the second peak in the amplitude histogram is generated by Ca2+ influx through N-type Ca2+ channels, causing a local elevation of internal Ca2+. The mSC amplitude in the positively skewed portion of the histogram varied over a wide range. A competitive blocker of acetylcholine (ACh) receptors, d-tubocurarine, reduced the amplitude of smaller mSCs in this range relatively more than that of larger mSCs, suggesting that this variation in the mSC amplitude is due to variable amounts of ACh released from synaptic vesicles. We suggest that Ca2+ influx through N-type Ca2+ channels preferentially induces release of vesicles with large ACh content.

Single-channel analysis of a K channel at basolateral membrane of rabbit proximal convoluted tubule

1988 ◽  
Vol 254 (1) ◽  
pp. F105-F113 ◽  
Author(s):  
L. Parent ◽  
J. Cardinal ◽  
R. Sauve
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
Proximal Convoluted Tubule ◽  
Time Interval ◽  
Distribution Analysis ◽  
Patch Clamp Technique ◽  
Bathing Medium ◽  
Patch Pipette ◽  
K Concentration ◽  
Interval Distribution

The basolateral membrane of the rabbit proximal convoluted tubule (PCT) is known to be largely permeable to K ions. The patch-clamp technique was used to investigate the molecular basis of this K permeability. At room temperature and with a high-K solution (127 mM) in both the bathing medium and the patch pipette, current jumps associated with an inward-rectifying channel could be detected in every active cell-attached experiment. When the K concentration in the pipette was changed from 200 to 5 mM KCl (NaCl replacement), the single-channel conductance for inward currents changed from 54 to 10 pS. The observed shift in the zero current potential measured as a function of the patch pipette K concentration could be fitted using the Goldman-Hodgkin-Katz equation with a permeability ratio PNa/PK = 0.06. The channel was found to be moderately voltage dependent (e-fold per 56 mV depolarization). For instance, the open-channel probability (Po) increased from 0.06 to 0.16 following a membrane depolarization from -50 to +50 mV. A time interval distribution analysis showed for the open state a dominant single time constant of 14 and 10 ms at 50 and -50 mV, respectively. Two time constants equal to 1 (flickering) and 26 ms at +50 mV and to 0.6 and 300 ms at -50 mV were obtained for the closed-state interval distribution. Based on this analysis, it was concluded that the decrease of Po at negative potentials was due more to the appearance of prolonged silent periods than from a change in the channel mean open time.

X-ray microanalysis of single cardiac myocytes frozen under voltage-clamp conditions

1989 ◽  
Vol 256 (2) ◽  
pp. H574-H583 ◽  
Author(s):  
M. F. Wendt-Gallitelli ◽  
G. Isenberg
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Microprobe Analysis ◽  
Ventricular Myocyte ◽  
Pulse Electron ◽  
Ca Current ◽  
X Ray ◽  
Freeze Dried ◽  
Patch Pipette ◽  
Intracellular Compartments ◽  
Junctional Sarcoplasmic Reticulum

By means of a patch pipette, an isolated ventricular myocyte was transferred into the taper of a silver holder covered by pioloform film. Once the cell was on the film, the cell was voltage clamped (pulses from -45 to +5 mV at 0.5 Hz). The amount of Ca entry was estimated from the Ca current. When contractility (cell shortening) was potentiated with either five pulses of 0.2 s or four pulses of 1 s, shock freezing was timed 116 or 816 ms after start of the clamp pulse. Electron micrographs from freeze-substituted cells revealed the good preservation of the intracellular compartments. The myocytes were cut at -150 degrees C, and the cryosections were freeze dried. In representative examples, the amount of Ca entry is compared with the subcellular Ca distribution as it is analyzed with energy dispersive X-ray microprobe analysis in cytoplasm, junctional sarcoplasmic reticulum (SR), mitochondria, and the subsarcolemmal space (sarcolemma, peripheral SR, fringe of cytosol).

scholarly journals Cyclic AMP-evoked oscillations of intracellular [Ca2+] in guinea-pig hepatocytes

1991 ◽  
Vol 275 (1) ◽  
pp. 277-280 ◽  
Author(s):  
T Capiod ◽  
J Noel ◽  
L Combettes ◽  
M Claret
Keyword(s):  
Angiotensin Ii ◽  
Cyclic Amp ◽  
Guinea Pig ◽  
Intracellular Perfusion ◽  
Beta Adrenoceptor ◽  
Adrenoceptor Agonist ◽  
Patch Pipette

The effects of the beta-adrenoceptor agonist isoprenaline and cyclic AMP (cAMP) on cytosolic free Ca2+ ([Ca2+]i) were studied in the single guinea-pig hepatocyte. In common with InsP3-dependent agonists such as noradrenaline or angiotensin II, isoprenaline (0.5-10 microM) and cAMP (50-100 mM, perfused into the cell via the patch-pipette), were able to generate fast and slow fluctuations of [Ca2+]i. Responses to isoprenaline and cAMP also were observed in the absence of external Ca2+. Isoprenaline-evoked [Ca2+]i rises were not blocked by the intracellular perfusion of heparin, suggesting that these fluctuations are independent of the binding of InsP3 to its receptor.

scholarly journals Oligomycin inhibits store-operated channels by a mechanism independent of its effects on mitochondrial ATP

1997 ◽  
Vol 324 (3) ◽  
pp. 971-980 ◽  
Author(s):  
Jwa Hwa CHO ◽  
M. BALASUBRAMANYAM ◽  
Galina CHERNAYA ◽  
Jeffrey P. GARDNER ◽  
Abraham AVIV ◽  
...  
Keyword(s):  
T Cells ◽  
Rank Order ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Inhibitory Effect ◽  
Jurkat T Cells ◽  
Ovary Cells ◽  
Atp Production ◽  
Patch Pipette ◽  
Pump Activity

Inhibitors of mitochondrial oxidative metabolism have been proposed to interfere with Ca2+ influx mediated by store-operated channels (SOC), secondary to their effects on ATP production. We assessed SOC activity by 45Ca2+ influx and fluorimetric measurements of free Ca2+ or Mn2+ quench in thapsigargin-treated Chinese hamster ovary cells and Jurkat T-cells, and additionally by electrophysiological measurements of the Ca2+-release-activated Ca2+ current (Icrac) in Jurkat T-cells. Various mitochondrial antagonists were confirmed to inhibit SOC. However, the following evidence supported the proposal that oligomycin, in particular, exerts an inhibitory effect on SOC in addition to its known actions on mitochondria and Na+-pump activity: (i) the concentrations of oligomycin required to inhibit SOC-mediated Ca2+ influx or Icrac (half-inhibitory concentration ∼2 μM) were nearly 50-fold higher than the concentrations that blocked mitochondrial ATP production; (ii) the rank order of potency of oligomycins A, B and C for decreasing SOC-mediated Ca2+ influx or Icrac differed from that known for inhibition of mitochondrial function; (iii) oligomycin blocked Icrac under voltage clamp and with intracellular Na+ and K+ concentrations fixed by dialysis from the patch pipette, arguing that the effect was not secondary to membrane polarization or pump activity; and (iv) fixing the cytosolic ATP concentration by dialysis from the patch pipette attenuated rotenone- but not oligomycin-mediated inhibition of Icrac. Oligomycin also blocked volume-activated Cl- currents, a profile common to some other known blockers of SOC that are not known mitochondrial inhibitors. These findings raise the possibility that oligomycin interacts directly with SOC, and thus may extend the known pharmacological profile for this type of Ca2+-influx pathway.

A 3,4-diaminopyridine-insensitive, Ca(2+)-independent transient outward K+ current in cardiac ventricular myocytes

1994 ◽  
Vol 266 (4) ◽  
pp. H1286-H1299 ◽  
Author(s):  
R. L. Martin ◽  
P. L. Barrington ◽  
R. E. Ten Eick
Keyword(s):  
Voltage Dependence ◽  
Ventricular Myocytes ◽  
Boltzmann Distribution ◽  
Time To Peak ◽  
Patch Pipette ◽  
Steady State Inactivation ◽  
Slope Factor ◽  
K Current ◽  
K Currents ◽  
Single Exponential

A previously unrecognized current that initially is not present and requires at least 25 min of intracellular access to develop can be found in approximately 75% of cardiac myocytes isolated from cat ventricle within 90 min after intracellular access is obtained with conventional suction patch pipette electrodes. We refer to this patch-duration-dependent (PDD) current as IK(PDD). IK(PDD) can be elicited with depolarizing test steps (Vt) ranging between -40 and +60 mV applied after a hyperpolarizing conditioning step to -140 mV for 200 ms from a holding potential of -40 mV. It shows an ohmic voltage dependence and appears to be an essentially pure K+ current. At Vt = 30 mV, the current is a time-dependent, transient current with a time to peak of 1.06 +/- 0.10 ms (n = 5) and a decay phase that can be fit to the sum of two decaying exponentials (tau f = 3.30 +/- 0.51 ms and tau s = 2.48 +/- 5.6 ms; n = 5). The voltage dependence of the steady-state inactivation can be fit to a single exponential Boltzmann distribution with a slope factor of 8.97 mV, and the voltage at which 50% of the channels are inactivated is -78 mV. The current can be blocked by 0.2 mM Ba2+ extracellularly applied or Cs+ intracellularly applied but is insensitive to 0.5 mM 3,4-diaminopyridine. These characteristics are unlike those for other known K+ currents. The lack of similarity between IK(PDD) and any currently documented cardiac K+ current suggests that IK(PDD) is either a previously undescribed K+ current or a modification of IK1 that makes it adopt an ohmic nature transiently, even in the presence of millimolar internal Mg2+.

InsP3 causes an increase in apical [Ca2+]i by activating two distinct current components in vertebrate olfactory receptor cells

1995 ◽  
Vol 73 (2) ◽  
pp. 862-866 ◽  
Author(s):  
D. Schild ◽  
F. W. Lischka ◽  
D. Restrepo
Keyword(s):  
Plasma Membrane ◽  
Calcium Imaging ◽  
Olfactory Receptor ◽  
Patch Clamp Technique ◽  
Olfactory Receptor Cells ◽  
Receptor Cells ◽  
Cation Current ◽  
Cation Conductance ◽  
Inositol 1,4,5 Trisphosphate ◽  
Patch Pipette

1. Effects of inositol-1,4,5-trisphosphate (InsP3) applied through a patch pipette to Xenopus laevis olfactory receptor cells (ORCs) were studied using the patch-clamp technique in conjunction with calcium imaging with fura-2. 2. InsP3 activated, first, a novel voltage-independent Ca2+ current (ICa) and, second, a nonselective cation current (Icat). 3. The activation of these currents occurred at different intracellular calcium concentrations, and the activation of either current led to a marked increase of [Ca2+]i in the dendritic knob. 4. The results suggest that InsP3 might act as a second messenger in vertebrate olfactory receptor cells by activating, through different mechanisms, a plasma membrane Ca2+ conductance (ICa) and a nonselective cation conductance (Icat).

Potentiation of a slow Ca(2+)-dependent K+ current by intracellular Ca2+ chelators in hippocampal CA1 neurons of rat brain slices

1995 ◽  
Vol 74 (6) ◽  
pp. 2225-2241 ◽  
Author(s):  
L. Zhang ◽  
P. Pennefather ◽  
A. Velumian ◽  
M. Tymianski ◽  
M. Charlton ◽  
...  
Keyword(s):  
Decay Time ◽  
Time Course ◽  
Brain Slices ◽  
Tetraacetic Acid ◽  
Pipette Solution ◽  
Ca1 Neurons ◽  
High Affinity ◽  
Hippocampal Ca1 ◽  
Patch Pipette ◽  
K Current

1. In hippocampal CA1 neurons of rat brain slices, a Ca(2+)-dependent slow afterhyperpolarization (sAHP) and underlying K+ current (IsAHP) are activated by Ca2+ influx and presumably reflect the time course of the intracellular Ca2+ signal produced by neuronal stimulation. We tested the hypothesis that when exogenous Ca2+ chelators become the predominant mobile Ca2+ buffer in the neuron, they alter the shape of intracellular Ca2+ signals responsible for IsAHP. The nature of this alteration provides insight into the mechanism of IsAHP generation. 2. Derivatives of 1,2-bis-[2-amino phenoxy] ethane N,N,N',N' tetraacetic acid (BAPTA) with different dissociation constants (KDS) for Ca2+ ranging from 0.15 to 7,000 microM were used to test this hypothesis. We also examined the effects of ethylene glycolbis (beta-aminoethyl either)-N,N,N',N'-tetraacetic acid (EGTA), which has a KD similar to that of BAPTA for Ca2+, but which binds and releases Ca2+ 100 times more slowly. When delivered to the cytoplasm by dialysis from a patch pipette, these chelators potentiated, inhibited, or had no effect on IsAHP depending on their concentration, affinity, and binding kinetics. 3. IsAHP decayed exponentially over much of its time course, with a half-decay time of 0.9 +/- 0.1 s (mean +/- SE, n = 22). Immediately after breakthrough into the whole cell configuration, there was an initial period of approximately 5 min during which IsAHP magnitude increased approximately 3.5-fold with no change in time course. Thereafter, the time course and amplitude of IsAHP were stable for > 45 min. 4. Addition of 1 mM of the high-affinity chelators 5,5'-dimethyl BAPTA or BAPTA to the pipette solution first increased the decay time of IsAHP 1.5-fold. However, within 10-15 min after break-through, the current was abolished. Addition of Ca2+ (0.1-1.0 mM) to the patch pipette containing the BAPTA derivatives reduced the ability of a given concentration of high-affinity chelator to inhibit IsAHP and also prolonged the period of IsAHP enhancement. A similar prolongation of the period of enhancement with even less attenuation of IsAHP was apparent with 0.1 mM 5,5'-dimethyl BAPTA and 0.1 mM Ca2+. 5. The intermediate-affinity chelator 4.4'-difluoro BAPTA (1 mM) prolonged the decay phase of the sAHP/IsAHP without attenuating the current. A twofold prolongation of IsAHP also was observed in neurons dialyzed with internal solution containing 3 mM EGTA and 0.3 mM Ca2+. Dialysis with 1 mM of the low-affinity chelators 2-amino-5-fluorophenol-N,N,O-triacetic acid (5-fluoro APTRA) or 5,5'-dinitro BAPTA had no apparent effect on IsAHP. All of the chelators that prolonged the decay phase of IsAHP also induced a rising phase such that a well-defined peak of IsAHP could be discerned at approximately 0.6 s after the end of the stimulus used to evoke the current. 6. Weak stimulation of muscarinic receptors selectively inhibits IsAHP. Thus the uncontaminated time course of IsAHP can be deduced by subtracting currents recorded before and after such muscarinic stimulation. With minimal exogenous buffer in the pipette (0.1 mM EGTA), the muscarinic-receptor-sensitive current exhibited a rising phase lasting approximately 300 ms and then decayed with a half-time of approximately 1 s. Both the rising and decay phases of the muscarinic-receptor-sensitive current were prolonged at least twofold by dialysis with BAPTA or 4,4'-difluoro BAPTA. Thus the effect of the chelators on the time course of IsAHP is not simply and artifact of inhibition of early components of the outward current. 7. The effects of BAPTA analogues on the time course of IsAHP are not due to changes in mobilization of intracellular Ca2+. External application of caffeine (10 mM), ryanodine (20 microM), dantrolene (20 microM), or thapsigargin (100 microM) had no effect on IsAHP recorded with the standard pipette solution or

scholarly journals Phosphatidylinositol-4,5-Biphosphate (PI(4,5)P2) Is Required for Rapid Endocytosis in Chromaffin Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-6
Author(s):  
Fouad Azizi
Keyword(s):  
Neurotransmitter Release ◽  
Chromaffin Cells ◽  
Inhibitory Effect ◽  
Adrenal Chromaffin Cells ◽  
Patch Clamp Recording ◽  
Vesicle Recycling ◽  
Patch Pipette ◽  
Cell Membrane Capacitance ◽  
Adrenal Chromaffin ◽  
Phosphatidylinositol 4

Objective. Phosphoinositides play a regulatory role in clathrin-mediated endocytosis. However, their involvement in clathrin-independent endocytosis termed rapid endocytosis (RE), which is the mode of vesicle recycling during neurotransmitter release by transient fusion (known as kiss-and-run), has not been investigated. Here, we used patch-clamp recording of whole-cell membrane capacitance in adrenal chromaffin cells (ACC) to monitor changes of RE kinetics in response to pharmacological alteration of phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) level by phenylarsine oxide (PAO) or antibody against phosphatidylinositol 4-kinase (AbPI4K). Results. We found that PAO and AbPI4K significantly abrogated RE kinetics. Infusion of PI(4,5)P2 through the patch pipette potentiated RE kinetics and reversed PAO- and AbPI4K-induced blockade of RE. Similarly, the application of the bifunctional thiol dithiothreitol (DTT) to PAO-treated cells completely prevented the inhibitory effect of PAO on RE. These findings indicate that PI(4,5)P2 is implicated in the signaling (mechanistic) process of RE in ACC.

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