PKC regulation of cardiac CFTR Cl− channel function in guinea pig ventricular myocytes

1998 ◽  
Vol 275 (1) ◽  
pp. C293-C302 ◽  
Author(s):  
Lisa M. Middleton ◽  
Robert D. Harvey
Keyword(s):  
Protein Kinase ◽  
Patch Clamp ◽  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Patch Clamp Technique ◽  
Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Perforated Patch Clamp

The role of protein kinase C (PKC) in regulating the protein kinase A (PKA)-activated Cl− current conducted by the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (cCFTR) was studied in guinea pig ventricular myocytes using the whole cell patch-clamp technique. Although stimulation of endogenous PKC with phorbol 12,13-dibutyrate (PDBu) alone did not activate this Cl− current, even when intracellular dialysis was limited with the perforated patch-clamp technique, activation of PKC did elicit a significant response in the presence of PKA-dependent activation of the current by the β-adrenergic receptor agonist isoproterenol. PDBu increased the magnitude of the Cl− conductance activated by a supramaximally stimulating concentration of isoproterenol by 21 ± 3.3% ( n = 9) when added after isoproterenol and by 36 ± 16% ( n= 14) when introduced before isoproterenol. 4α-Phorbol 12,13-didecanoate, a phorbol ester that does not activate PKC, did not mimic these effects. Preexposure to chelerythrine or bisindolylmaleimide, two highly selective inhibitors of PKC, significantly reduced the magnitude of the isoproterenol-activated Cl− current by 79 ± 7.7% ( n = 11) and 52 ± 10% ( n = 8), respectively. Our results suggest that although acute activation of endogenous PKC alone does not significantly regulate cCFTR Cl− channel activity in native myocytes, it does potentiate PKA-dependent responses, perhaps most dramatically demonstrated by basal PKC activity, which may play a pivotal role in modulating the function of these channels.

Altered beta-adrenergic and muscarinic response of CFTR Cl- current in dialyzed cardiac myocytes

1995 ◽  
Vol 268 (5) ◽  
pp. H1795-H1802
Author(s):  
S. I. Zakharov ◽  
R. D. Harvey
Keyword(s):  
Patch Clamp ◽  
Muscarinic Receptor ◽  
Ventricular Myocytes ◽  
Receptor Activation ◽  
Current Response ◽  
Patch Clamp Technique ◽  
Beta Adrenergic ◽  
Perforated Patch ◽  
Clamp Technique ◽  
Perforated Patch Clamp

Autonomic regulation of the cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- current was studied in isolated guinea pig ventricular myocytes using various configurations of the whole cell patch-clamp technique. When currents were recorded using the conventional patch-clamp technique, it was possible to continue to activate the Cl- current on repeated exposure to isoproterenol (Iso) for up to 60 min after initiating dialysis. However, there was significant rundown of the magnitude of the Cl- current response to the maximally stimulating concentrations of Iso. In addition, the concentration of Iso that produced half-maximal activation of the Cl- current (K1/2) increased with time. Conversely, the K1/2 for acetylcholine inhibition of the Iso-activated current decreased with time. When currents were recorded using the perforated patch-clamp technique, the sensitivity to both beta-adrenergic- and muscarinic-receptor stimulation was stable. Immediately after initiation of dialysis with the conventional patch-clamp technique, the sensitivity to Iso was nearly identical to that determined using the perforated patch-clamp technique. However, the initial sensitivity to muscarinic-receptor activation was significantly greater. These results indicate that cell dialysis associated with conventional patch-clamp techniques not only results in a time-dependent rundown of current amplitude, but it also significantly alters the concentration dependence of beta-adrenergic and muscarinic-receptor regulation of ion channel function.

Force measurements from voltage-clamped guinea pig ventricular myocytes

1990 ◽  
Vol 258 (2) ◽  
pp. H452-H459 ◽  
Author(s):  
N. Shepherd ◽  
M. Vornanen ◽  
G. Isenberg
Keyword(s):  
Guinea Pig ◽  
Time Course ◽  
Ventricular Myocytes ◽  
Isometric Force ◽  
Contractile Force ◽  
Patch Clamp Technique ◽  
Thin Glass ◽  
Tonic Component ◽  
Time To Peak ◽  
Whole Cell Patch Clamp

We describe the first observations of isolated mammalian guinea pig ventricular myocytes that combine measurements of contractile force with the voltage-clamp method. The myocytes were attached by poly-L-lysine to the beveled ends of a pair of thin glass rods having a compliance of 0.76 m/N. The contractile force of a cell caused a 1- to 3-microm displacement of the rods; the motion of which was converted to an output voltage by phototransistors. By the use of the whole cell patch-clamp technique, the cells were depolarized at 1 Hz with 200-ms-long clamp pulses from -45 to +5 mV (35 degrees C, 3.6 mM CaCl2). Isometric force began after a latency of 7 +/- 2 ms, peaked at 93 +/- 21 ms, and relaxed (90%) at 235 +/- 63 ms. The time course of force was always faster than that of isotonic shortening (time to peak 154 +/- 18 ms). With 400-ms-long depolarizations, a tonic component was recorded as either sustained force or sustained shortening that decayed on repolarization. Substitution of Ca by Sr in the bath increased the inward current through Ca channels but slowed down the time course of force development. The results are consistent with the hypothesis that activator calcium derives mainly from internal stores and that Ca release needs Ca entry through channels.

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

Gramicidin perforated patch-clamp technique reveals glycine-gated outward chloride current in dissociated nucleus solitarii neurons of the rat

1994 ◽  
Vol 72 (3) ◽  
pp. 1103-1108 ◽  
Author(s):  
J. S. Rhee ◽  
S. Ebihara ◽  
N. Akaike
Keyword(s):  
Patch Clamp ◽  
Hill Coefficient ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Perforated Patch ◽  
Clamp Technique ◽  
Outward Currents ◽  
Patch Technique ◽  
Perforated Patch Clamp

1. The inhibitory response of exogenously applied glycine was investigated in freshly dissociated rat nucleus tractus solitarii neurons under whole cell configuration using new perforated patch-clamp technique termed "gramicidin perforated patch technique," which maintains intact intracellular Cl- concentrations. 2. Using the gramicidin perforated patch technique, at a holding potential (VH) of -45 mV, glycine induced outward currents in a concentration-dependent manner with a EC50 of 4.0 x 10(-5) M and at a Hill coefficient of 1.5. In contrast, using the nystatin perforated patch technique, glycine induced inward currents at the same VH in a concentration-dependent manner with an EC50 of 4.9 x 10(-5) M and at a Hill coefficient of 1.2. 3. The glycine-induced outward currents were blocked by strychnine in a concentration dependent manner with an IC50 of 2.2 x 10(-8) M. The blockade was competitive. 4. The current-voltage relationship for the 10(-5) M glycine response showed a clear outward rectification. 5. Ten-fold change of extracellular Cl- with a large impermeable anion resulted in a 65 mV shift of the reversal potential of glycine-induced currents (EGly), indicating that the membrane behaves like a Cl- electrode in the presence of glycine. 6. The intracellular Cl- activity calculated from the EGly ranged from 7.3 to 18.2 mM, with a mean value of 13.3 mM. 7. The values of EGly in the individual neurons were significantly negative to the resting membrane potentials, suggesting the existence of active transport of Cl-.

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.

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