scholarly journals Analysis of neuronal Ca2+ handling properties by combining perforated patch clamp recordings and the added buffer approach

2020 ◽  
Author(s):  
Simon Hess ◽  
Christophe Pouzat ◽  
Lars Paeger ◽  
Andreas Pippow ◽  
Peter Kloppenburg
Keyword(s):  
Patch Clamp ◽  
Intracellular Signaling ◽  
Whole Cell ◽  
Perforated Patch ◽  
Cellular Processes ◽  
Wide Range ◽  
Intracellular Ca ◽  
Precise Quantification ◽  
Perforated Patch Clamp ◽  
Whole Cell Recordings

AbstractCa2+ functions as an important intracellular signal for a wide range of cellular processes. These processes are selectively activated by controlled spatiotemporal dynamics of the free cytosolic Ca2+. Intracellular Ca2+ dynamics are regulated by numerous cellular parameters. Here, we established a new way to determine neuronal Ca2+ handling properties by combining the ‘added buffer’ approach (Neher and Augustine, 1992) with perforated patch-clamp recordings (Horn and Marty, 1988). Since the added buffer approach typically employs the standard whole-cell configuration for concentration-controlled Ca2+ indicator loading, it only allows for the reliable estimation of the immobile fraction of intracellular Ca2+ buffers. Furthermore, crucial components of intracellular signaling pathways are being washed out during prolonged whole-cell recordings, leading to cellular deterioration. By combining the added buffer approach with perforated patch-clamp recordings, these issues are circumvented, allowing the precise quantification of the cellular Ca2+ handling properties, including immobile as well as mobile Ca2+ buffers.

scholarly journals Whole-cell and Perforated Patch-clamp Recordings from Acutely-isolated Murine Sino-atrial Node Cells

BIO-PROTOCOL ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Qadeer Aziz ◽  
Muriel Nobles ◽  
Andrew Tinker
Keyword(s):  
Patch Clamp ◽  
Whole Cell ◽  
Perforated Patch ◽  
Perforated Patch Clamp

Carbachol induces K+, Cl-, and nonselective cation conductances in T84 cells: a perforated patch-clamp study

1992 ◽  
Vol 263 (4) ◽  
pp. C780-C787 ◽  
Author(s):  
D. C. Devor ◽  
M. E. Duffey
Keyword(s):  
Patch Clamp ◽  
T84 Cells ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Inward Current ◽  
Perforated Patch ◽  
Ionic Conductances ◽  
Inward Currents ◽  
K Current ◽  
Perforated Patch Clamp

We used the perforated patch-clamp technique to examine cell membrane ionic conductances in isolated cells of the human colonic secretory cell line, T84, during exposure to the muscarinic agonist carbachol. Carbachol (100 microM) induced both outward and inward currents when the patch pipette contained a normal intracellular-like solution, the bath contained a normal extracellular-like solution, and the cells were intermittently voltage clamped between K+ and Cl- equilibrium potentials. The outward current was identified as a K+ current that averaged 483 +/- 95 pA, while the inward current averaged 152 +/- 29 pA (n = 15). The outward and inward currents oscillated with a synchronous frequency of 0.036 +/- 0.006 Hz; however, the onset of the K+ current occurred an average of 457 +/- 72 ms before the onset of the inward current. When the pipette contained a high-NaCl solution, the bath contained a Na(+)-gluconate solution, and the cells were intermittently voltage clamped between Cl- and Na+ equilibrium potentials, carbachol induced both Cl- and nonselective cation currents. The Cl- current averaged 455 +/- 73 pA, while the nonselective cation current, averaged 336 +/- 54 pA (n = 14). No difference was observed in the onset of these two currents. These results indicate that carbachol induces three separate ionic conductances in T84 cells. We used the whole cell patch-clamp technique in a previous study of these cells [D. C. Devor, S. M. Simasko, and M. E. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990] and found that carbachol induced only an oscillating membrane K+ conductance. Thus some unidentified component of the carbachol-sensitive signal transduction pathway is diffusible and may be lost during whole cell patch clamping.

Modulation of ICa-L by α1-adrenergic stimulation in rat ventricular myocytes

2005 ◽  
Vol 83 (11) ◽  
pp. 1015-1024 ◽  
Author(s):  
Shetuan Zhang ◽  
Jijin Lin ◽  
Yuji Hirano ◽  
Masayasu Hiraoka
Keyword(s):  
Protein Kinase ◽  
Patch Clamp ◽  
Kinase Inhibitor ◽  
Ventricular Myocytes ◽  
Adrenergic Stimulation ◽  
Pipette Solution ◽  
Rat Ventricular Myocytes ◽  
Perforated Patch ◽  
Intracellular Ca ◽  
Perforated Patch Clamp

We found when L-type calcium current (ICa-L) was recorded with the perforated patch-clamp method in rat ventricular myocytes that bath application of phenylephrine (with propranolol) evoked a biphasic response characterized by an initial transient suppression followed by a sustained potentiation. The transient suppression occurred 30–60 s after phenylephrine perfusion and reached peak inhibition at approximately 2 min. The biphasic modulation of ICa-L was also elicited by methoxamine, and the effects of phenylephrine were blocked by prazosin, indicating that the responses were mediated through α1-adrenoceptors. Pretreatment of cells with H7 (100 µmol/L), a broad-spectrum protein kinase inhibitor that inhibits both protein kinase C and A, eliminated potentiation but did not affect transient suppression. The transient suppression occurred concurrently with the acceleration of the fast component of ICa-L inactivation. Depletion of intracellular Ca2+ stores by ryanodine plus caffeine or thapsigargin eliminated the transient suppression. When ICa-L was recorded with whole-cell patch-clamp and with 0.05 mmol/L EGTA in the pipette solution to allow intracellular Ca2+ to fluctuate, phenylephrine evoked a transient suppression as in the perforated patch recordings. Heparin, a specific blocker of IP3 (inositol 1,4,5-trisphosphate) receptors, eliminated the phenylephrine-induced transient suppression of ICa-L when added to the pipette solution. Intensive chelation of intracellular Ca2+ by 5 mmol/L BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) in the pipette solution also eliminated the phenylephrine-induced transient suppression of ICa-L. We conclude that transient increase in the concentration of intracellular calcium ([Ca2+]i) caused by Ca2+ release from intracellular stores underlies the transient suppression of ICa-L, whereas the potentiation of ICa-L is a result of activation of protein kinases.Key words: Ca2+ mobilization, IP3, Ca2+-induced inactivation of Ca2+ current, perforated patch-clamp.

scholarly journals Ca2+ release from the sarcoplasmic reticulum is required for sustained TRPM4 activity in cerebral artery smooth muscle cells

2010 ◽  
Vol 299 (2) ◽  
pp. C279-C288 ◽  
Author(s):  
Albert L. Gonzales ◽  
Gregory C. Amberg ◽  
Scott Earley
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Patch Clamp ◽  
Smooth Muscle Cells ◽  
Cerebral Artery ◽  
Perforated Patch ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Trisphosphate Receptor ◽  
Perforated Patch Clamp

The melastatin transient receptor potential (TRP) channel TRPM4 is a critical regulator of vascular smooth muscle cell membrane potential and contractility. Activation of the channel is Ca2+-dependent, but prolonged exposure to high (>1 μM) levels of intracellular Ca2+ causes rapid (within ∼2 min) desensitization of TRPM4 currents under conventional whole cell and inside-out patch-clamp conditions. The goal of the present study was to establish a novel method to record sustained TRPM4 currents in smooth muscle cells under near-physiological conditions. Using the amphotericin B-perforated patch-clamp technique, we recorded and characterized sustained (up to 30 min) transient inward cation currents (TICCs) in freshly isolated cerebral artery myocytes. In symmetrical cation solutions, TICCs reversed at 0 mV and had an apparent unitary conductance of 25 pS. Replacement of extracellular Na+ with the nonpermeable cation N-methyl-d-glucamine abolished the current. TICC activity was attenuated by the TRPM4 blockers fluflenamic acid and 9-phenanthrol. Selective silencing of TRPM4 expression using small interfering RNA diminished TICC activity, suggesting that the molecular identity of the responsible ion channel is TRPM4. We used the perforated patch-clamp method to test the hypothesis that TRPM4 is activated by intracellular Ca2+ signaling events. We found that TICC activity is independent of Ca2+ influx and ryanodine receptor activity but is attenuated by sarco(endo)plasmic reticulum Ca2+-ATPase inhibition and blockade of inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release from the sarcoplasmic reticulum. Our findings suggest that TRPM4 channels in cerebral artery myocytes are regulated by Ca2+ release from inositol 1,4,5-trisphosphate receptor on the sarcoplasmic reticulum.

Membrane Potential of CA3 Hippocampal Pyramidal Cells During Postnatal Development

2003 ◽  
Vol 90 (5) ◽  
pp. 2964-2972 ◽  
Author(s):  
Roman Tyzio ◽  
Anton Ivanov ◽  
Cristophe Bernard ◽  
Gregory L. Holmes ◽  
Yehezkiel Ben-Ari ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Postnatal Development ◽  
Developmental Change ◽  
Hippocampal Slices ◽  
Pyramidal Cells ◽  
Input Resistance ◽  
Whole Cell ◽  
Perforated Patch ◽  
Ca3 Pyramidal Cells ◽  
Whole Cell Recordings

A depolarized resting membrane potential has long been considered to be a universal feature of immature neurons. Despite the physiological importance, the underlying mechanisms of this developmental phenomenon are poorly understood. Using perforated-patch, whole cell, and cell-attached recordings, we measured the membrane potential in CA3 pyramidal cells in hippocampal slices from postnatal rats. With gramicidin perforated-patch recordings, membrane potential was –44 ± 4 (SE) mV at postnatal days P0–P2, and it progressively shifted to –67 ± 2 mV at P13–15. A similar developmental change of the membrane potential has been also observed with conventional whole cell recordings. However, the value of the membrane potential deduced from the reversal potential of N-methyl-d-aspartate channels in cell-attached recordings did not change with age and was –77 ± 2 mV at P2 and –77 ± 2 mV at P13–14. The membrane potential measured using whole cell recordings correlated with seal and input resistance, being most depolarized in neurons with high, several gigaohms, input resistance and low seal resistance. Simulations revealed that depolarized values of the membrane potential in whole cell and perforated-patch recordings could be explained by a shunt through the seal contact between the pipette and membrane. Thus the membrane potential of CA3 pyramidal cells appears to be strongly negative at birth and does not change during postnatal development.

Alteration of microtubule polymerization modulates arteriolar vasomotor tone

1999 ◽  
Vol 277 (1) ◽  
pp. H100-H106 ◽  
Author(s):  
Steven H. Platts ◽  
Jeff C. Falcone ◽  
William T. Holton ◽  
Michael A. Hill ◽  
Gerald A. Meininger
Keyword(s):  
Time Course ◽  
Contractile Function ◽  
Vasomotor Tone ◽  
Microtubule Depolymerization ◽  
Similar Time ◽  
Cellular Processes ◽  
Endothelial Denudation ◽  
Wide Range ◽  
Intracellular Ca ◽  
Cytoskeletal Elements

Microtubules are important cytoskeletal elements that have been shown to play a major role in many cellular processes because of their mechanical properties and/or their participation in various cell signaling pathways. We tested the hypothesis that depolymerization of microtubules would alter vascular smooth muscle (VSM) tone and hence contractile function. In our studies, isolated cremaster arterioles exhibited significant vasoconstriction that developed over a 20- to 40-min period when they were treated with microtubule depolymerizing drugs colchicine (10 μM), nocodazole (10 μM), or demecolcine (10 μM). Immunofluorescent labeling of microtubules in cultured rat VSM revealed that both colchicine and nocodazole caused microtubule depolymerization over a similar time course. The vasoconstriction was maintained over a wide range of intraluminal pressures (30–170 cmH2O). The increased tone was not affected by endothelial denudation, suggesting that it was due to an effect on VSM. Microtubule depolymerization with demecolcine or colchicine had no effect on VSM intracellular Ca2+ concentration ([Ca2+]i). These data indicate that microtubules significantly interact with processes leading to the expression of vasomotor tone. The mechanism responsible for the effect of microtubules on vasomotor tone appears to be independent of both the endothelium and an increase in VSM [Ca2+]i.

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