Antagonistic Modulation of a Hyperpolarization-Activated Cl– Current in Aplysia Sensory Neurons by SCPB and FMRFamide

2003 ◽  
Vol 90 (2) ◽  
pp. 586-598 ◽  
Author(s):  
Ned Buttner ◽  
Steven A. Siegelbaum
Keyword(s):  
Sensory Neurons ◽  
Reversal Potential ◽  
Outward Current ◽  
Cell Voltage ◽  
High Concentration ◽  
Pipette Solution ◽  
Whole Cell ◽  
Whole Cell Recording ◽  
Type K ◽  
Cell Recording

Whole cell voltage-clamp recordings from Aplysia mechanosensory neurons obtained from the pleural ganglion were used to investigate the actions on membrane currents of the neuropeptides SCPB and FMRFamide. At the start of whole cell recording, SCPB typically evoked an inward current at a holding potential of –40 mV, due to the cAMP-mediated closure of the S-type K+ channel, whereas FMRFamide evoked an outward current, due to the opening of the S-type K+ channels mediated by 12-lipoxygenase metabolites of arachidonic acid. However, after several minutes of whole cell recording with a high concentration of chloride in the whole cell patch pipette solution, the responses to SCPB and FMRF-amide at –40 mV were inverted; SCPB evoked an outward current, whereas FMRFamide and YGGFMRFamide evoked inward currents. Ion substitution experiments and reversal potential measurements revealed that these responses were due to the opposing regulation of a Cl– current, whose magnitude was greatly enhanced by dialysis with the high Cl–-containing pipette solution. SCPB inhibited this Cl– current through production of cAMP and activation of PKA. YGGFMRFamide activated this Cl– current by stimulating a cGMP-activated phosphodiesterase that hydrolyzed cAMP. Thus a cAMP-dependent Cl– current undergoes antagonistic modulation by two neuropeptides in Aplysia sensory neurons.

Perforated-patch recording does not enhance effect of 3-isobutyl-1-methylxanthine on cardiac calcium current

1994 ◽  
Vol 266 (6) ◽  
pp. C1619-C1627 ◽  
Author(s):  
A. Kawamura ◽  
G. M. Wahler
Keyword(s):  
Ventricular Myocytes ◽  
Cell Voltage ◽  
Rapid Decline ◽  
Response Curves ◽  
Whole Cell ◽  
Perforated Patch ◽  
Cell Responses ◽  
Whole Cell Recording ◽  
Cell Recording ◽  
Beta Adrenergic Agonist

Conventional whole cell voltage-clamp recording results in washout of the cardiac Ca2+ current (ICa) response to the beta-adrenergic agonist isoproterenol (Iso), for reasons which are not clear. When dose-response curves for the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) were compared using perforated-patch vs. conventional whole cell recording in guinea pig ventricular myocytes, the conventional whole cell IBMX responses were unexpectedly larger than the perforated-patch responses. Furthermore, during conventional whole cell recording the response to repeated application of Iso declined rapidly, whereas the IBMX response initially increased and then declined. When pipette [Ca2+] was increased to 10(-7) M, conventional whole cell responses to 300 microM IBMX and 10(-9) M Iso were identical to perforated-patch responses. Thus loss of the Iso response during conventional whole cell recording seems to not be solely due to a washout of some constituent of the adenosine 3',5'-cyclic monophosphate pathway. We suggest that unphysiological intracellular [Ca2+] enhances the relative PDE activity and that this contributes to the rapid decline of the Iso response and the initial enhancement of the IBMX response.

Odor-induced currents in Xenopus olfactory receptor cells measured with perforated-patch recording

1995 ◽  
Vol 74 (1) ◽  
pp. 479-483 ◽  
Author(s):  
A. B. Zhainazarov ◽  
B. W. Ache
Keyword(s):  
Olfactory Receptor ◽  
Reversal Potential ◽  
Olfactory Receptor Neurons ◽  
Whole Cell ◽  
Time To Peak ◽  
Olfactory Receptor Cells ◽  
Whole Cell Recording ◽  
Cell Recording ◽  
Receptor Neurons ◽  
Induced Currents

1. Odor-evoked currents were recorded in Xenopus laevis olfactory receptor neurons (ORNs) by the use of conventional, as well as nystatin and gramicidin-perforated, whole cell recording. The odor-evoked current ran down quickly in conventional, but not in perforated, whole cell recording. All three types of recording gave similar values for the amplitude, latency, time-to-peak, recovery time, and reversal potential of the odor-evoked current. 2. A secondary Cl current comprised a significant part of the odor-evoked current (55-65%). ECl measured by gramicidin perforation, which does not alter [Cl-]i, was -2.3 +/- 5.0 (SE) mV, indicating that these neurons maintain a high [Cl-]i and that the secondary Cl current plays an excitatory role in olfactory transduction.

Hydrogen peroxide activates glibenclamide-sensitive K+ channels in LLC-PK1 cells

1997 ◽  
Vol 272 (2) ◽  
pp. C737-C743 ◽  
Author(s):  
D. M. Filipovic ◽  
W. B. Reeves
Keyword(s):  
Patch Clamp ◽  
Ion Selectivity ◽  
Oxidant Stress ◽  
Reversal Potential ◽  
Cell Voltage ◽  
Atp Depletion ◽  
Pipette Solution ◽  
Membrane Hyperpolarization ◽  
Whole Cell ◽  
K Channels

Oxidant-induced damage has been implicated in the pathogenesis of several forms of cellular injury. The present study employed patch-clamp methods to determine if oxidant stress leads to activation of plasma membrane K+ channels in the renal epithelial LLC-PK1 cell line. Exposure of cells to H2O2 (0.1 to 5 mM) induced a rapid (within 5-10 min), dose-dependent membrane hyperpolarization. Perforated patch whole cell voltage-clamp studies were performed to determine the ion selectivity of the currents underlying this H2O2-induced cellular hyperpolarization. H2O2 (5 mM) produced a sixfold increase in the whole cell conductance. The reversal potential of the H2O2-induced current was consistent with a K+-selective conductance. This current was blocked almost completely by 5 mM barium and 500 microM glibenclamide but only partially by 15 mM tetraethylammonium. Exposure of LLC-PK1 cells to 5 mM H2O2 reduced cell ATP content by 70%. To evaluate more directly the role of ATP depletion in the activation of K+ channels, conventional whole cell patch-clamp studies were performed. Inclusion of ATP in the pipette solution prevented H2O2-induced activation of the K+ conductance. These findings indicate that H2O2 activates an ATP-sensitive, Ca2+-independent K+ conductance in LLC-PK1 cells.

Whole cell recording of sugar-induced currents in LLC-PK1 cells

1990 ◽  
Vol 258 (2) ◽  
pp. C234-C242 ◽  
Author(s):  
C. Smith-Maxwell ◽  
E. Bennett ◽  
J. Randles ◽  
G. A. Kimmich
Keyword(s):  
Transport System ◽  
Sugar Transport ◽  
Reversal Potential ◽  
Electrical Current ◽  
Bathing Medium ◽  
Whole Cell ◽  
Whole Cell Recording ◽  
Cell Recording ◽  
Coupling Stoichiometry ◽  
Induced Currents

Gigaohm-seal whole cell recording techniques were used to monitor function of the Na(+)-coupled sugar transport system in LLC-PK1 cells. The currents coupled to sugar transport were identified as those that are induced by the presence of 10 mM alpha-methylglucoside (AMG) in either the extracellular or intracellular compartment and were inhibited by addition of 320-800 microM phlorizin to the extracellular bathing medium. The sugar-induced currents are small, 15-20 pA, but of the expected magnitude as determined from the known kinetic parameters for Na(+)-coupled sugar transport in LLC-PK1 cells. The phlorizin-sensitive currents are Na+ dependent and can be studied under conditions in which the net Na+ and sugar flux (and consequently the Na+ electrical current) is in either the inward or outward direction. The reversal potential of the sugar-induced currents measured under conditions with high Na+ and AMG concentrations inside the cell is close to values predicted from thermodynamic principles, assuming a coupling stoichiometry of 2 Na+: 1 sugar for the transport system. The reversal potential of the sugar-induced currents with high extracellular Na+ and AMG is not equal to the predicted value, but it is of the polarity expected for inward-imposed solute gradients. Reasons for the observed discrepancy between observed and calculated values are discussed.

Relationship between changes of glomus cell current and neural response of rat carotid body

1995 ◽  
Vol 74 (5) ◽  
pp. 2077-2086 ◽  
Author(s):  
P. M. Cheng ◽  
D. F. Donnelly
Keyword(s):  
Carotid Body ◽  
Outward Current ◽  
Membrane Resistance ◽  
Glomus Cell ◽  
Nerve Activity ◽  
Whole Cell ◽  
Glomus Cells ◽  
Perforated Patch ◽  
Whole Cell Recording ◽  
Cell Recording

1. Mature rat carotid bodies were harvested and sinus nerve activity was recorded in vitro during superfusion with Ringer saline. Membrane currents of glomus cells were simultaneously recorded using conventional whole cell or perforated-patch whole cell recording. Presumptive glomus cells were identified by the presence of a rapidly activated, voltage-dependent outward current above a threshold of -20 mV. 2. Outward current of presumptive glomus cells was inhibited by tetraethylammonium chloride (TEA) (20 mM) and by verapamil (5-10 microM), consistent with previous studies in which isolated glomus cells were used. Somal capacitance, calculated from the current transient following a step hyperpolarization, was 7.47 +/- 0.54 (SE) pF (n = 52). Membrane resistance for perforated-patch recordings was 820 +/- 187 M omega. 3. In perforated-patch recordings, brief periods of hypoxia (30-45 s) caused a marked increase in nerve activity to 21.6 +/- 2.7 times baseline spiking frequency (n = 59) but no significant change in membrane resistance or outward current. No change in holding current was detected, although the low amplifier gain precluded high-resolution measurement. Similar results were obtained using conventional whole cell recording, except that outward current significantly decreased during hypoxia but failed to recover in the immediate posthypoxia period. 4. TEA (20 mM) rapidly inhibited outward current to 55 +/- 7% (n = 15) of predrug current, but nerve activity only slightly increased to 2.0 +/- 0.3 times baseline spike frequency (n = 15). Brief anoxia (40 s in duration) in the presence of TEA evoked a brisk increase in nerve activity to 30 +/- 13 times baseline frequency (n = 3), demonstrating that organ function was not blocked by TEA. 5. Charybdotoxin (10 nM) significantly reduced outward current by 12.1 +/- 3.0% (n = 11) but did not significantly alter nerve activity, holding current, or membrane resistance. Apamin (100 nM) did not significantly affect nerve activity, membrane resistance, or holding current. Outward current decreased by 11.4 +/- 6.1% (n = 13). 6. These results show a dissociation between changes in glomus cell voltage-gated outward currents and changes in afferent nerve activity. This suggests that modulation of glomus cell K+ current by hypoxia is not the primary step in initiating the nerve response to hypoxia in the rat carotid body.

scholarly journals Diverse K+ channels in primary human T lymphocytes.

1992 ◽  
Vol 99 (5) ◽  
pp. 771-793 ◽  
Author(s):  
S C Lee ◽  
D I Levy ◽  
C Deutsch
Keyword(s):  
T Lymphocytes ◽  
Outward Current ◽  
Cell Voltage ◽  
Whole Cell ◽  
K Channels ◽  
Voltage Gated ◽  
T Lymphocyte Proliferation ◽  
Cell Recording ◽  
Gated Channel ◽  
K Current

We used patch clamp techniques to identify and characterize a variety of K+ channels in primary human peripheral T lymphocytes. The most common channel observed in cell-attached configuration was voltage gated and inactivating. In ensemble averages, the kinetics of its activation and inactivation were similar to those of the whole-cell, voltage-gated K+ current described previously (Cahalan, M. D., K. G. Chandy, T. E. DeCoursey, and S. Gupta. 1985. J. Physiol. [Lond.]. 358:197-237; Deutsch, C., D. Krause, and S. C. Lee. 1986. J. Physiol. [Lond.]. 372:405-423), suggesting that this channel underlies the major portion of the outward current in lymphocytes. A small fraction of the time, this or another very similar channel was observed to inactivate significantly more slowly. Another channel type observed in cell-attached recording was seen less frequently and was transient in its appearance. This channel has a unitary conductance of approximately 10 pS, similar to the voltage-gated channel, but its voltage-independent gating, lack of inactivation, and different kinetic parameters showed it to be distinct. In whole-cell recording there is often a significant plateau current during sustained depolarization. Experiments using whole-cell and excised outside-out configurations indicate that at least part of this residual current is carried by K+ and, as opposed to the predominant voltage-gated current, is charybdotoxin insensitive. These findings are consistent with evidence that implicates charybdotoxin-sensitive and -insensitive components in T lymphocyte proliferation and volume regulation.

Nitric oxide mediates outward potassium currents in opossum esophageal circular smooth muscle

1996 ◽  
Vol 270 (6) ◽  
pp. G932-G938 ◽  
Author(s):  
J. Jury ◽  
K. R. Boev ◽  
E. E. Daniel
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Circular Muscle ◽  
Outward Current ◽  
Equilibrium Potential ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Circular Smooth Muscle ◽  
Outward Currents

Single smooth muscle cells from the opossum body circular muscle were isolated and whole cell currents were characterized by the whole cell patch-clamp technique. When the cells were held at -50 mV and depolarized to 70 mV in 20-mV increments, initial small inactivating inward currents were evoked (-30 to 30 mV) followed by larger sustained outward currents. Depolarization from a holding potential of -90 mV evoked an initial fast inactivating outward current sensitive to 4-aminopyridine but not to high levels of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The outward currents reversed near K+ equilibrium potential and were abolished when KCl was replaced by CsCl in the pipette solution. The sustained outward current was inhibited by quinine and cesium. High EGTA in the pipette solution reduced but did not abolish the sustained outward currents, suggesting that both Ca(2+)-dependent and -independent currents were evoked. The nitric oxide (NO)-releasing agents Sin-1 and sodium nitroprusside increased outward K+ currents. High levels of EGTA in the pipette solution abolished the increase in outward current induced by Sin-1. The presence of cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum (SR) Ca2+ pump, blocked the effects of NO-releasing agents. We conclude that NO release activates K+ outward currents in opossum esophagus circular muscle, which may depend on Ca2+ release from the SR stores.

Whole cell recording from neurons in slices of reptilian and mammalian cerebral cortex

1989 ◽  
Vol 30 (3) ◽  
pp. 203-210 ◽  
Author(s):  
Mark G. Blanton ◽  
Joseph J. Lo Turco ◽  
Arnold R. Kriegstein
Keyword(s):  
Cerebral Cortex ◽  
Whole Cell ◽  
Whole Cell Recording ◽  
Cell Recording

scholarly journals Knockouts reveal overlapping functions of M2 and M4 muscarinic receptors and evidence for a local glutamatergic circuit within the laterodorsal tegmental nucleus

2012 ◽  
Vol 108 (10) ◽  
pp. 2751-2766 ◽  
Author(s):  
Kristi A. Kohlmeier ◽  
Masaru Ishibashi ◽  
Jürgen Wess ◽  
Martha E. Bickford ◽  
Christopher S. Leonard
Keyword(s):  
Acetylcholine Receptors ◽  
Brain Slices ◽  
Cholinergic Neurons ◽  
Muscarinic Acetylcholine Receptors ◽  
Feedback Signal ◽  
Laterodorsal Tegmental Nucleus ◽  
Membrane Hyperpolarization ◽  
Whole Cell ◽  
Whole Cell Recording ◽  
Cell Recording

Cholinergic neurons in the laterodorsal tegmental (LDT) and peduncolopontine tegmental (PPT) nuclei regulate reward, arousal, and sensory gating via major projections to midbrain dopamine regions, the thalamus, and pontine targets. Muscarinic acetylcholine receptors (mAChRs) on LDT neurons produce a membrane hyperpolarization and inhibit spike-evoked Ca2+ transients. Pharmacological studies suggest M2 mAChRs are involved, but the role of these and other localized mAChRs (M1--M4) has not been definitively tested. To identify the underlying receptors and to circumvent the limited receptor selectivity of available mAChR ligands, we used light- and electron-immunomicroscopy and whole cell recording with Ca2+ imaging in brain slices from knockout mice constitutively lacking either M2, M4, or both mAChRs. Immunomicroscopy findings support a role for M2 mAChRs, since cholinergic and noncholinergic LDT and pedunculopontine tegmental neurons contain M2-specific immunoreactivity. However, whole cell recording revealed that the presence of either M2 or M4 mAChRs was sufficient, and that the presence of at least one of these receptors was required for these carbachol actions. Moreover, in the absence of M2 and M4 mAChRs, carbachol elicited both direct excitation and barrages of spontaneous excitatory postsynaptic potentials (sEPSPs) in cholinergic LDT neurons mediated by M1 and/or M3 mAChRs. Focal carbachol application to surgically reduced slices suggest that local glutamatergic neurons are a source of these sEPSPs. Finally, neither direct nor indirect excitation were knockout artifacts, since each was detected in wild-type slices, although sEPSP barrages were delayed, suggesting M2 and M4 receptors normally delay excitation of glutamatergic inputs. Collectively, our findings indicate that multiple mAChRs coordinate cholinergic outflow from the LDT in an unexpectedly complex manner. An intriguing possibility is that a local circuit transforms LDT muscarinic inputs from a negative feedback signal for transient inputs into positive feedback for persistent inputs to facilitate different firing patterns across behavioral states.

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