scholarly journals Slow inactivation of L-type calcium current distorts the measurement of L- and T-type calcium current in Purkinje myocytes.

1993 ◽  
Vol 102 (5) ◽  
pp. 859-869 ◽  
Author(s):  
N B Datyner ◽  
I S Cohen
Keyword(s):  
Patch Clamp ◽  
Calcium Current ◽  
Slow Inactivation ◽  
Total Calcium ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Voltage Curve ◽  
Voltage Dependent ◽  
Method Of Measurement

We have examined slow inactivation of L-type calcium current in canine Purkinje myocytes with the whole cell patch clamp technique. Slow inactivation is voltage dependent. It is negligible at -50 mV but can inactivate more than half of available iCaL at -10 mV. There are two major consequences of this slow inactivation. First, standard protocols for the measurement of T-type current can dramatically overestimate its contribution to total calcium current, and second, the position and steepness of the inactivation versus voltage curve for iCaL will depend on the method of measurement. Given the widespread attempts to identify calcium current components and characterize them biophysically, an important first step should be to determine the extent of slow inactivation of calcium current in each preparation.

scholarly journals Inhibition of cardiac Na+ currents by isoproterenol

1990 ◽  
Vol 258 (4) ◽  
pp. H977-H982 ◽  
Author(s):  
B. Schubert ◽  
A. M. Vandongen ◽  
G. E. Kirsch ◽  
A. M. Brown
Keyword(s):  
Patch Clamp ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Na Channel ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Na Currents ◽  
Voltage Dependent

The mechanism by which the beta-adrenergic agonist isoproterenol (ISO) modulates voltage-dependent cardiac Na+ currents (INa) was studied in single ventricular myocytes of neonatal rat using the gigaseal patch-clamp technique. ISO inhibited INa reversibly, making the effect readily distinguishable from the monotonic decrease of INa caused by the shift in gating that customarily occurs during whole cell patch-clamp experiments (E. Fenwick, A. Marty, and E. Neher, J. Physiol. Lond. 331: 599-635, 1982; and J. M. Fernandez, A. P. Fox, and S. Krasne, J. Physiol. Lond. 356: 565-585, 1984). The inhibition was biphasic, having fast and slow components, and was voltage-dependent, being more pronounced at depolarized potentials. In whole cell experiments the membrane-permeable adenosine 3',5'-cyclic monophosphate (cAMP) congener 8-bromo-cAMP reduced INa. In cell-free inside-out patches with ISO present in the pipette, guanosine 5'-triphosphate (GTP) applied to the inner side of the membrane patch inhibited single Na+ channel activity. This inhibition could be partly reversed by hyperpolarizing prepulses. The nonhydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) greatly reduced the probability of single Na+ channel currents in a Mg2(+)-dependent manner. We propose that ISO inhibits cardiac Na+ channels via the guanine nucleotide binding, signal-transducing G protein that acts through both direct (membrane delimited) and indirect (cytoplasmic) pathways.

Whole-cell patch-clamp analysis of voltage-dependent calcium conductances in cultured embryonic rat hippocampal neurons

1989 ◽  
Vol 61 (3) ◽  
pp. 467-477 ◽  
Author(s):  
D. E. Meyers ◽  
J. L. Barker
Keyword(s):  
Patch Clamp ◽  
Hippocampal Neurons ◽  
Calcium Currents ◽  
Tetraacetic Acid ◽  
Whole Cell ◽  
Inward Current ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Cells Cultured ◽  
In Cells

1. Voltage-dependent calcium currents in embryonic (E18) hippocampal neurons cultured for 1-14 days were investigated using the whole-cell patch-clamp technique. 2. Calcium currents were isolated by removing K+ from both the internal and external solutions. In most recordings the external solution contained tetrodotoxin, tetraethylammonium ions, and low concentrations of Na+, whereas the internal solution contained the large cations and anions, N-methyl-D-glucamine and methanesulphonate, and an adenosine 5'-triphosphate (ATP) regenerating system (Forscher and Oxford, 1985) to retard “run-down” of Ca currents. 3. Under these conditions, the sustained inward current triggered during depolarizing steps was enhanced when extracellular [Ca2+] ([Ca2+]0) was raised from 2 to 10 mM and abolished when [Ca2+]0 was lowered to 0.1 mM or by addition of Co2+ ions. These results indicate that the inward current was carried primarily by Ca2+ ions and was designated ICa. This current may be comparable to the “high-voltage-activated” Ca current described in other preparations. 4. In cells cultured for 1-3 days, ICa was small or absent (less than 20 pA for cells 1 day in culture and less than 80 pA for cells 3 days in culture). Although ICa decayed considerably during depolarizing steps, there was little evidence of the transient calcium current (T current) that was recorded in approximately 40% of cells cultured longer than 6 days. Maximal (i.e., the largest) ICa increased from 20 to 80 pA in 1- to 3-day cells to 150–450 pA in cells cultured for longer than 6 days. 5. The decay of ICa elicited by depolarizations from holding potentials of -60 mV or more negative was usually greatest for the maximal ICa. Replacement of extracellular Ca2+ (4 mM) with Ba2+ (2 mM) resulted in a substantial decrease in the extent of decay of ICa and a shift of the I-V relation in the hyperpolarizing direction. 6. Qualitative data obtained from experiments in which different levels of internal Ca2+ buffering were employed demonstrated that, on average, the decay of ICa was reduced as the capacity and/or rate of buffering was increased. The mean decay of ICa in cells buffered with 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was 9 +/- 7 (SD) %, (n = 12) and 25 +/- 12%, (n = 12) for cells buffered with the same concentration of ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Baro-excited neurons in the caudal ventrolateral medulla (CVLM) recorded using the whole-cell patch-clamp technique

2011 ◽  
Vol 35 (5) ◽  
pp. 500-506 ◽  
Author(s):  
Naoki Oshima ◽  
Hiroo Kumagai ◽  
Kamon Iigaya ◽  
Hiroshi Onimaru ◽  
Akira Kawai ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Ventrolateral Medulla ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Caudal Ventrolateral Medulla ◽  
Clamp Technique ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

scholarly journals Neutrophil Secretion Induced by an Intracellular Ca2+Rise and Followed by Whole-Cell Patch-Clamp Recordings Occurs Without any Selective Mobilization of Different Granule Populations

2006 ◽  
Vol 2006 ◽  
pp. 1-7 ◽  
Author(s):  
Daniel Granfeldt ◽  
Olle Harbecke ◽  
Åse Björstad ◽  
Anna Karlsson ◽  
Claes Dahlgren
Keyword(s):  
Patch Clamp ◽  
Human Neutrophils ◽  
Lag Phase ◽  
Measuring Time ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
High Concentrations ◽  
Kinetics Of

We have investigated calcium-induced secretion in human neutrophils, using a whole-cell patch-clamp technique. Mobilization of subcellular granules to the cell membrane was followed as the change in membrane capacitance (△Cm). Both the magnitude and the kinetics of the response differed between low and high concentrations of Ca2+. A sustained secretion following a short lag phase was induced by high concentrations of Ca2+(100μM and higher). A stable plateau was reached after 5–7 minutes at△Cmvalues corresponding to values expected after all specific as well as azurophil granules have been mobilized. Capacitance values of the same magnitude could be obtained also at lower Ca2+concentrations, but typically no stable plateau was reached within the measuring time. In contrast to previous studies, we were unable to detect any pattern of secretion corresponding to a distinct submaximal response or selective mobilization of granule subsets specified by their Ca2+-sensitivity.

Membrane properties of mesopontine cholinergic neurons studied with the whole-cell patch-clamp technique: implications for behavioral state control

1992 ◽  
Vol 68 (4) ◽  
pp. 1359-1372 ◽  
Author(s):  
A. Kamondi ◽  
J. A. Williams ◽  
B. Hutcheon ◽  
P. B. Reiner
Keyword(s):  
Patch Clamp ◽  
Cholinergic Neurons ◽  
Membrane Properties ◽  
Type I ◽  
Type Ii ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Type Iii ◽  
Clamp Technique ◽  
Whole Cell Patch Clamp

1. The whole-cell patch-clamp technique was used to study the membrane properties of identified cholinergic and noncholinergic laterodorsal tegmental neurons in slices of rat brain maintained in vitro. 2. On the basis of their expression of the transient outward potassium current IA and the transient inward calcium current IT, three classes of neurons were observed: type I neurons exhibited a large IT; type II neurons exhibited a prominent IA; and type III neurons exhibited both IA and IT. 3. Combining intracellular deposition of biocytin with NADPH diaphorase histochemistry revealed that the vast majority of type III neurons were cholinergic, whereas only a minority of type I and type II neurons were cholinergic. Thus mesopontine cholinergic neurons possess intrinsic ionic currents capable of inducing burst firing. 4. Delineation of the intrinsic membrane properties of identified mesopontine cholinergic neurons, in concert with recent results regarding the responses of these neurons to neurotransmitter agents, has led us to present a unifying and mechanistic hypothesis of brain stem cholinergic function in the control of behavioral states.

Neuropeptide Y reduces calcium current and inhibits acetylcholine release in nodose neurons via a pertussis toxin-sensitive mechanism

1990 ◽  
Vol 63 (6) ◽  
pp. 1499-1507 ◽  
Author(s):  
J. W. Wiley ◽  
R. A. Gross ◽  
Y. X. Lu ◽  
R. L. Macdonald
Keyword(s):  
Patch Clamp ◽  
Neuropeptide Y ◽  
Pertussis Toxin ◽  
Calcium Current ◽  
Calcium Currents ◽  
High Threshold ◽  
Time Constants ◽  
Whole Cell ◽  
Peak Current ◽  
Whole Cell Patch Clamp

1. The effect of neuropeptide Y (NPY) on voltage-dependent calcium currents was studied in acutely dissociated rat vagal afferent (nodose) neurons by the use of both intracellular single-electrode and whole-cell patch-clamp techniques. 2. Nodose neurons exhibited three calcium current components similar to the transient low-threshold (T), slowly inactivating high-threshold (L), and the transient high-threshold (N) currents previously described in dorsal root ganglion neurons (Nowycky et al. 1985). The characteristics of calcium current components were similar for the two recording techniques except that the inactivation time constants (tau i) were two- to threefold larger at 22 degrees C (whole-cell patch clamp) than at 35 degrees C (single-electrode voltage clamp). 3. NPY (0.1-100 nM, ED50 4 nM) produced a concentration-dependent reduction in calcium currents with the use of both recording techniques. NPY (100 nM) had no effect on T and L currents but reduced the combined N/L current 31 +/- 6% in 47% of the cells tested. Current traces were also analyzed by multiexponential curve fitting to determine amplitudes and inactivation time constants (tau i). NPY selectively reduced the amplitude of the curve-fitted N current component 45 +/- 8% but had no effect on any of the tau i. The effect of NPY to reduce calcium current was blocked in the presence of gadolinium (1 microM), a putative N channel antagonist. Pretreatment of cultures with pertussis toxin (PTX) (100 ng/ml) for 16-24 h blocked the effect of NPY. 4. NPY reduced the peak current without changing the voltage dependence of the peak current-voltage relation.(ABSTRACT TRUNCATED AT 250 WORDS)

GABA-Mimetic Actions of Withania somnifera on Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis in Mice

2013 ◽  
Vol 41 (05) ◽  
pp. 1043-1051 ◽  
Author(s):  
Hua Yin ◽  
Dong Hyu Cho ◽  
Soo Joung Park ◽  
Seong Kyu Han
Keyword(s):  
Patch Clamp ◽  
Receptor Antagonist ◽  
Withania Somnifera ◽  
Substantia Gelatinosa ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Inward Currents

The plant Withania somnifera (WS), also known as Ashwagandha, has been used widely in traditional medicine systems in India and Nepal (Ayurveda), and has been accepted to cure various ailments. In this study, the whole-cell patch clamp technique was performed to examine the mechanism of action of WS on the SG neurons of the Vc from mouse brainstem slices. In whole-cell patch clamp mode, methanol extract of Withania somnifera (mWS) induced short-lived and repeatable inward currents in all SG neurons tested (31.3±8.51 pA, n = 7) using a high chloride pipette solution. The mWS-induced inward currents were concentration dependent and maintained in the presence of tetrodotoxin (TTX), a voltage gated Na + channel blocker, CNQX, a non-NMDA glutamate receptor antagonist, AP5, an NMDA receptor antagonist and strychnine, a glycine receptor antagonist. The mWS induced currents were blocked by picrotoxin, a GABAA receptor antagonist. These results show that mWS has an inhibitory effects on SG neurons of the Vc through GABAA receptor-mediated activation of chloride ion channels, indicating that mWS contains compounds with sedative effects on the central nervous system. These results also suggest that mWS may be a potential target for modulating orofacial pain processing.

scholarly journals Thapsigargin-induced Ca2+ mobilization in acutely isolated mouse lacrimal acinar cells is dependent on a basal level of Ins(1,4,5)P3 and is inhibited by heparin

1994 ◽  
Vol 299 (1) ◽  
pp. 37-40 ◽  
Author(s):  
P M Smith ◽  
D V Gallacher
Keyword(s):  
Patch Clamp ◽  
Atpase Activity ◽  
Receptor Antagonist ◽  
Acinar Cells ◽  
Cell Types ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Subsequent Exposure ◽  
Lacrimal Acinar Cells

The tumour-promoting agent thapsigargin has been shown to inhibit the microsomal Ca(2+)-ATPase and cause Ca2+ mobilization in a variety of cell types including exocrine acinar cells [Bird, Obie and Putney (1992) J. Biol. Chem. 267, 18382-18386]. When applied to acutely isolated lacrimal acinar cells, thapsigargin caused a slow biphasic activation of both the Ca(2+)-dependent K+ and Cl- currents measured using the whole-cell patch-clamp technique. If the only action of thapsigargin is to inhibit sequestration into Ca2+ pools, then Ca2+ mobilization following exposure to thapsigargin indicates that there is a significant ‘leak’ of Ca2+ into the cytoplasm, which is normally countered by Ca(2+)-ATPase activity. In the present study, we introduced the Ins(1,4,5)P3 receptor antagonist heparin (200 micrograms/ml) into lacrimal acinar cells via the patch-clamp pipette. Following a 5 min preincubation in the presence of heparin, neither acetylcholine (1 microM) nor thapsigargin (1 microM) caused any significant increase in either Ca(2+)-dependent current. Caffeine has been shown to suppress basal Ins(1,4,5)P3 levels in exocrine acinar cells [Toescu, O'Neill, Petersen and Eisner (1992) J. Biol. Chem. 267, 23467-23470]. Preincubation with caffeine (10 mM) also inhibited the response to subsequent exposure to thapsigargin. These data suggest that, in acutely isolated lacrimal cells, the source of the Ca2+ leak which gives rise to Ca2+ mobilization following inhibition of Ca2+ re-uptake by thapsigargin is Ca2+ release, from Ins(1,4,5)P3-dependent Ca2+ pools, caused by resting Ins(1,4,5)P3 levels.

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