scholarly journals Voltage-gated and Ca(2+)-activated K+ channels in intact human T lymphocytes. Noninvasive measurements of membrane currents, membrane potential, and intracellular calcium.

1995 ◽  
Vol 105 (6) ◽  
pp. 765-794 ◽  
Author(s):  
J A Verheugen ◽  
H P Vijverberg ◽  
M Oortgiesen ◽  
M D Cahalan
Keyword(s):  
Membrane Potential ◽  
T Lymphocytes ◽  
Reversal Potential ◽  
Resting Membrane Potential ◽  
Ca Channel ◽  
Ca Channels ◽  
Channel Activity ◽  
Intact Cells ◽  
Human T Lymphocytes ◽  
High K

Voltage-gated n-type K(V) and Ca(2+)-activated K+ [K(Ca)] channels were studied in cell-attached patches of activated human T lymphocytes. The single-channel conductance of the K(V) channel near the resting membrane potential (Vm) was 10 pS with low K+ solution in the pipette, and 33 pS with high K+ solution in the pipette. With high K+ pipette solution, the channel showed inward rectification at positive potentials. K(V) channels in cell-attached patches of T lymphocytes inactivated more slowly than K(V) channels in the whole-cell configuration. In intact cells, steady state inactivation at the resting membrane potential was incomplete, and the threshold for activation was close to Vm. This indicates that the K(V) channel is active in the physiological Vm range. An accurate, quantitative measure for Vm was obtained from the reversal potential of the K(V) current evoked by ramp stimulation in cell-attached patches, with high K+ solution in the pipette. This method yielded an average resting Vm for activated human T lymphocytes of -59 mV. Fluctuations in Vm were detected from changes in the reversal potential. Ionomycin activates K(Ca) channels and hyperpolarizes Vm to the Nernst potential for K+. Elevating intracellular Ca2+ concentration ([Ca2+]i) by ionomycin opened a 33-50-pS channel, identified kinetically as the CTX-sensitive IK-type K(Ca) channel. The Ca2+ sensitivity of the K(Ca) channel in intact cells was determined by measuring [Ca2+]i and the activity of single K(Ca) channels simultaneously. The threshold for activation was between 100 and 200 nM; half-maximal activation occurred at 450 nM. At concentrations > 1 microM, channel activity decreased. Stimulation of the T-cell receptor/CD3 complex using the mitogenic lectin, PHA, increased [Ca2+]i, and increased channel activity and current amplitude resulting from membrane hyperpolarization.

scholarly journals Cardiac calcium channels in planar lipid bilayers. L-type channels and calcium-permeable channels open at negative membrane potentials.

1988 ◽  
Vol 92 (1) ◽  
pp. 27-54 ◽  
Author(s):  
R L Rosenberg ◽  
P Hess ◽  
R W Tsien
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Detailed Comparison ◽  
Membrane Potentials ◽  
Bay K 8644 ◽  
Ca Channel ◽  
Ca Channels ◽  
Channel Activity ◽  
Intact Cells ◽  
Free System

Planar lipid bilayer recordings were used to study Ca channels from bovine cardiac sarcolemmal membranes. Ca channel activity was recorded in the absence of nucleotides or soluble enzymes, over a range of membrane potentials and ionic conditions that cannot be achieved in intact cells. The dihydropyridine-sensitive L-type Ca channel, studied in the presence of Bay K 8644, was identified by a detailed comparison of its properties in artificial membranes and in intact cells. L-type Ca channels in bilayers showed voltage dependence of channel activation and inactivation, open and closed times, and single-channel conductances in Ba2+ and Ca2+ very similar to those found in cell-attached patch recordings. Open channels were blocked by micromolar concentrations of external Cd2+. In this cell-free system, channel activity tended to decrease during the course of an experiment, reminiscent of Ca2+ channel "rundown" in whole-cell and excised-patch recordings. A purely voltage-dependent component of inactivation was observed in the absence of Ca2+ stores or changes in intracellular Ca2+. Millimolar internal Ca2+ reduced unitary Ba2+ influx but did not greatly increase the rate or extent of inactivation or the rate of channel rundown. In symmetrical Ba2+ solutions, unitary conductance saturated as the Ba2+ concentration was increased up to 500 mM. The bilayer recordings also revealed activity of a novel Ca2+-permeable channel, termed "B-type" because it may contribute a steady background current at negative membrane potentials, which is distinct from L-type or T-type Ca channels previously reported. Unlike L-type channels, B-type channels have a small unitary Ba2+ conductance (7 pS), but do not discriminate between Ba2+ and Ca2+, show no obvious sensitivity to Bay K 8644, and do not run down. Unlike either L- or T-type channels, B-type channels did not require a depolarization for activation and displayed mean open times of greater than 100 ms.

Evidence for a Ca-activated inwardly rectifying K channel in human macrophages

1989 ◽  
Vol 257 (1) ◽  
pp. C77-C85 ◽  
Author(s):  
E. K. Gallin
Keyword(s):  
Membrane Potential ◽  
Single Channel ◽  
Reversal Potential ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Base Line ◽  
Channel Activity ◽  
Human Macrophages ◽  
Excised Patches ◽  
Single Channel Currents

Cell-attached patch studies of cultured human macrophages demonstrate that exposure to ionomycin induces inward-rectifying single-channel currents that differ from the voltage-dependent 28 pS inward-rectifying K currents previously described in these cells (J. Membr. Biol. 103: 55-66, 1988). With 150 mM KCl in the electrode and NaCl Hanks' solution in the bath, the ionomycin-induced single-channel conductance for inward currents was 37 pS, and the reversal potential was 57 mV. Channel activity was often associated with a shift in the base-line current level indicating that the cell membrane potential hyperpolarized. The ability of ionomycin to induce channel activity depended on extracellular [Ca] supporting the view that the channels were gated by calcium. Ionomycin-induced channels were permeable to K, relatively impermeable to Cl or Na, exhibited bursting kinetics, and had no apparent voltage dependence. Barium (3 mM in the patch electrode) did not significantly block the ionomycin-induced channel at rest but blocked channel activity when the patch was hyperpolarized beyond the resting membrane potential. Exposure of macrophages to platelet-activating factor, which is known to increase intracellular [Ca] [( Ca]i) (J. Cell Biol. 103: 439-450, 1986), also transiently induced channel activity. In excised patches with 3 microM [Ca]i bursting inward-rectifying channels with a 41 pS conductance were noted that probably correspond to the ionomycin-induced channels present in cell-attached patches. Increasing [Ca]i from 10(-8) to 3 x 10(-6) M induced inward-rectifying channel activity in previously quiescent excised patches.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of synaptically mediated fast and slow inhibitory processes in piriform cortex in an in vitro slice preparation

1988 ◽  
Vol 59 (5) ◽  
pp. 1352-1376 ◽  
Author(s):  
G. F. Tseng ◽  
L. B. Haberly
Keyword(s):  
Membrane Potential ◽  
Piriform Cortex ◽  
Reversal Potential ◽  
Pyramidal Cells ◽  
Membrane Depolarization ◽  
Resting Membrane Potential ◽  
Excitatory Postsynaptic Potential ◽  
Pentobarbital Sodium ◽  
Postsynaptic Potential ◽  
Conductance Increase

1. Intracellular recordings were obtained from anatomically verified layer II pyramidal cells in slices from rat piriform cortex cut perpendicular to the surface. 2. Responses to afferent and association fiber stimulation at resting membrane potential consisted of a depolarizing potential followed by a late hyperpolarizing potential (LHP). Membrane polarization by current injection revealed two components in the depolarizing potential: an initial excitatory postsynaptic potential (EPSP) followed at brief latency by an inhibitory postsynaptic potential (IPSP) that inverted with membrane depolarization and truncated the duration of the EPSP. 3. The early IPSP displayed the following characteristics suggesting mediation by gamma-aminobutyric acid (GABA) receptors linked to Cl- channels: associated conductance increase, sensitivity to increases in internal Cl- concentration, blockage by picrotoxin and bicuculline, and potentiation by pentobarbital sodium. The reversal potential was in the depolarizing direction with respect to resting membrane potential so that the inhibitory effect was exclusively via current shunting. 4. The LHP had an associated conductance increase and a reversal potential of -90 mV in normal bathing medium that shifted according to Nernst predictions for a K+ potential with changes in external K+ over the range 4.5-8 mM indicating mediation by the opening of K+ channels and ruling out an electrogenic pump origin. 5. Lack of effect of bath-applied 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) or internally applied ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) on the LHP and failure of high amplitude, direct membrane depolarization to evoke a comparable potential, argue against endogenous mediation of the LHP by a Ca2+ activated K+ conductance [gK(Ca)]. However, an apparent endogenously mediated gK(Ca) with a duration much greater than the LHP was observed in a low percent of layer II pyramidal cells. Lack of effect of 8-Br-cAMP also indicates a lack of dependence of the LHP on cAMP. 6. Other characteristics of the LHP that were demonstrated include: a lack of blockage by GABAA receptor antagonists, a probable voltage sensitivity (decrease in amplitude in the depolarizing direction), and an apparent brief onset latency (less than 10 ms) when the early IPSP was blocked by picrotoxin. The LHP was unaffected by pentobarbital sodium when the early IPSP was blocked by picrotoxin. 7. Both the LHP and early IPSP were blocked by low Ca2+/high Mg2+, consistent with disynaptic mediation.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of membrane potential by an acetylcholine-activated potassium current in trout atrial myocytes

2007 ◽  
Vol 292 (1) ◽  
pp. R388-R395 ◽  
Author(s):  
Cristina E. Molina ◽  
Hans Gesser ◽  
Anna Llach ◽  
Lluis Tort ◽  
Leif Hove-Madsen
Keyword(s):  
Membrane Potential ◽  
Action Potential ◽  
Ventricular Myocytes ◽  
Reversal Potential ◽  
Membrane Potentials ◽  
Resting Membrane Potential ◽  
Atrial Myocytes ◽  
Atrial Tissue ◽  
Isolated Cardiac Myocytes ◽  
Inwardly Rectifying

Application of the current-clamp technique in rainbow trout atrial myocytes has yielded resting membrane potentials that are incompatible with normal atrial function. To investigate this paradox, we recorded the whole membrane current ( Im) and compared membrane potentials recorded in isolated cardiac myocytes and multicellular preparations. Atrial tissue and ventricular myocytes had stable resting potentials of −87 ± 2 mV and −83.9 ± 0.4 mV, respectively. In contrast, 50 out of 59 atrial myocytes had unstable depolarized membrane potentials that were sensitive to the holding current. We hypothesized that this is at least partly due to a small slope conductance of Im around the resting membrane potential in atrial myocytes. In accordance with this hypothesis, the slope conductance of Im was about sevenfold smaller in atrial than in ventricular myocytes. Interestingly, ACh increased Im at −120 mV from 4.3 pA/pF to 27 pA/pF with an EC50 of 45 nM in atrial myocytes. Moreover, 3 nM ACh increased the slope conductance of Im fourfold, shifted its reversal potential from −78 ± 3 to −84 ± 3 mV, and stabilized the resting membrane potential at −92 ± 4 mV. ACh also shortened the action potential in both atrial myocytes and tissue, and this effect was antagonized by atropine. When applied alone, atropine prolonged the action potential in atrial tissue but had no effect on membrane potential, action potential, or Im in isolated atrial myocytes. This suggests that ACh-mediated activation of an inwardly rectifying K+ current can modulate the membrane potential in the trout atrial myocytes and stabilize the resting membrane potential.

The generation of electric currents in cardiac fibers by Na/Ca exchange

1979 ◽  
Vol 236 (3) ◽  
pp. C103-C110 ◽  
Author(s):  
L. J. Mullins
Keyword(s):  
Membrane Potential ◽  
Duty Cycle ◽  
Action Potentials ◽  
Reversal Potential ◽  
Resting Membrane Potential ◽  
Electrochemical Gradient ◽  
Ca Current ◽  
Cardiac Action ◽  
Cardiac Fiber ◽  
Cardiac Fibers

The presence of a detectable Ca current during the excitation of a cardiac fiber implies that the Ca lost during the resting interval of the duty cycle must also be detectable. Ca outward movement appears to be effected by Na/Ca exchange when more Na enters than Ca leaves per cycle, thus making the mechanism electrogenic. Since Na/Ca exchange can move Ca either inward or outward depending on the direction of the electrochemical gradient for Na, a potential exists where there is no electric current generated by the Na/Ca exchange mechanism, i.e., a reversal potential ER. Cardiac fibers appear to have a reversal potential that is about midway between their resting membrane potential and their plateau. Carrier currents both inward and outward are therefore generated during cardiac action potentials. The implications of the conditions stated above are explored.

Ca2+-activated Cl− current in cultured myenteric neurons from murine proximal colon

2003 ◽  
Vol 284 (4) ◽  
pp. C839-C847 ◽  
Author(s):  
Sok Han Kang ◽  
Pieter Vanden Berghe ◽  
Terence K. Smith
Keyword(s):  
Membrane Potential ◽  
Channel Blocker ◽  
Reversal Potential ◽  
Outward Current ◽  
Proximal Colon ◽  
Time Dependent ◽  
Equilibrium Potential ◽  
Resting Membrane Potential ◽  
Myenteric Neurons ◽  
Whole Cell Patch Clamp

Whole cell patch-clamp recordings were made from cultured myenteric neurons taken from murine proximal colon. The micropipette contained Cs+ to remove K+ currents. Depolarization elicited a slowly activating time-dependent outward current ( I tdo), whereas repolarization was followed by a slowly deactivating tail current ( I tail). I tdo and I tail were present in ∼70% of neurons. We identified these currents as Cl− currents ( I Cl), because changing the transmembrane Cl− gradient altered the measured reversal potential ( E rev) of both I tdo and I tail with that for I tailshifted close to the calculated Cl− equilibrium potential ( E Cl). I Cl are Ca2+-activated Cl− current [ I Cl(Ca)] because they were Ca2+dependent. E Cl, which was measured from the E rev of I Cl(Ca) using a gramicidin perforated patch, was −33 mV. This value is more positive than the resting membrane potential (−56.3 ± 2.7 mV), suggesting myenteric neurons accumulate intracellular Cl−. ω-Conotoxin GIVA [0.3 μM; N-type Ca2+ channel blocker] and niflumic acid [10 μM; known I Cl(Ca) blocker], decreased the I Cl(Ca). In conclusion, these neurons have I Cl(Ca) that are activated by Ca2+entry through N-type Ca2+ channels. These currents likely regulate postspike frequency adaptation.

Membrane depolarization in PC-12 cells during hypoxia is regulated by an O2-sensitive K+ current

1996 ◽  
Vol 271 (2) ◽  
pp. C658-C665 ◽  
Author(s):  
W. H. Zhu ◽  
L. Conforti ◽  
M. F. Czyzyk-Krzeska ◽  
D. E. Millhorn
Keyword(s):  
Membrane Potential ◽  
Reversal Potential ◽  
Outward Current ◽  
Cell Voltage ◽  
Membrane Depolarization ◽  
Resting Membrane Potential ◽  
Current Clamp ◽  
Pc 12 Cells ◽  
Current Voltage ◽  
K Current

The effects of hypoxia on K+ current (IK), resting membrane potential, and cytosolic free Ca2+ in rat pheochromocytoma (PC-12) cells were studied. Whole cell voltage- and current-clamp experiments were performed to measure IK and membrane potential, respectively. Cytosolic free Ca2+ level was measured using the Ca(2+)-sensitive fluorescent dye fura 2. Depolarizing voltage steps to +50 mV from a holding potential of -90 mV elicited a slowly inactivating, tetraethylammonium chloride-sensitive, and Ca(2+)-insensitive IK that was reversibly inhibited by reduced O2 tension. Graded reduction in PO2 (from 150 to 0 mmHg) induced a graded inhibition of O2-sensitive IK [IK(O2)] up to 46% at 0 mmHg. Moreover, hypoxia induced a 19-mV membrane depolarization and a twofold increase in cytosolic free Ca2+. In Ca(2+)-free condition, inhibition of IK(O2) induced an 8-mV depolarization, suggesting that inhibition of IK(O2) was responsible for initiating depolarization. The effect of reduced PO2 on the current-voltage relationship showed a reduction of outward current and a 14-mV shift in the reversal potential comparable with the amount of depolarization measured in current clamp experiments. Neither Ca(2+)-activated IK nor inwardly rectifying IK are responsible for the hypoxia-induced depolarization. In conclusion, PC-12 cells express an IK(O2), inhibition of which leads to membrane depolarization and increased intracellular Ca2+, making the PC-12 clonal cell line a useful model for studying the molecular and biophysical mechanisms that mediate O2 chemosensitivity.

Volatile Anesthetics Induce Caspase-dependent, Mitochondria-mediated Apoptosis in Human T Lymphocytes In Vitro 

2005 ◽  
Vol 102 (6) ◽  
pp. 1147-1157 ◽  
Author(s):  
Torsten Loop ◽  
David Dovi-Akue ◽  
Michael Frick ◽  
Martin Roesslein ◽  
Lotti Egger ◽  
...  
Keyword(s):  
Membrane Potential ◽  
T Lymphocytes ◽  
Cytochrome C ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Volatile Anesthetics ◽  
Human T Lymphocytes ◽  
Induced Apoptosis

Background Volatile anesthetics modulate lymphocyte function during surgery, and this compromises postoperative immune competence. The current work was undertaken to examine whether volatile anesthetics induce apoptosis in human T lymphocytes and what apoptotic signaling pathway might be used. Methods Effects of sevoflurane, isoflurane, and desflurane were studied in primary human CD3 T lymphocytes and Jurkat T cells in vitro. Apoptosis and mitochondrial membrane potential were assessed using flow cytometry after green fluorescent protein-annexin V and DiOC6-fluorochrome staining. Activity and proteolytic processing of caspase 3 was measured by cleaving of the fluorogenic effector caspase substrate Ac-DEVD-AMC and by anti-caspase-3 Western blotting. Release of mitochondrial cytochrome c was studied after cell fractionation using anti-cytochrome c Western blotting and enzyme-linked immunosorbent assays. Results Sevoflurane and isoflurane induced apoptosis in human T lymphocytes in a dose-dependent manner. By contrast, desflurane did not exert any proapoptotic effects. The apoptotic signaling pathway used by sevoflurane involved disruption of the mitochondrial membrane potential and release of cytochrome c from mitochondria to the cytosol. In addition, the authors observed a proteolytic cleavage of the inactive p32 procaspase 3 to the active p17 fragment, increased caspase-3-like activity, and cleavage of the caspase-3 substrate poly-ADP-ribose-polymerase. Sevoflurane-induced apoptosis was blocked by the general caspase inhibitor Z-VAD.fmk. Death signaling was not mediated via the Fas/CD95 receptor pathway because neither anti-Fas/CD95 receptor antagonism nor FADD deficiency or caspase-8 deficiency were able to attenuate sevoflurane-mediated apoptosis. Conclusion Sevoflurane and isoflurane induce apoptosis in T lymphocytes via increased mitochondrial membrane permeability and caspase-3 activation, but independently of death receptor signaling.

Molecular Insights into Regulation of L-Type Ca Channel Function

Physiology ◽  
1991 ◽  
Vol 6 (6) ◽  
pp. 277-281 ◽  
Author(s):  
P Lory ◽  
G Varadi ◽  
A Schwartz
Keyword(s):  
Structure Function ◽  
Splice Variants ◽  
Ca Channel ◽  
Gene Products ◽  
Ca Channels ◽  
Channel Activity ◽  
Excitable Cells ◽  
Multiple Gene ◽  
Channel Function ◽  
Voltage Dependent

The diversity of voltage-dependent Ca channels is well documented. How excitable cells produce their specific Ca channel activity is being approached by structure-function studies. The implications of multiple gene products, splice variants, and subunit assembly in Ca channel function are updated in this review.

Membrane Properties Underlying Patterns of GABA-Dependent Action Potentials in Developing Mouse Hypothalamic Neurons

2001 ◽  
Vol 86 (3) ◽  
pp. 1252-1265 ◽  
Author(s):  
Yu-Feng Wang ◽  
Xiao-Bing Gao ◽  
Anthony N. van den Pol
Keyword(s):  
Membrane Potential ◽  
Action Potentials ◽  
Brain Slices ◽  
Reversal Potential ◽  
Membrane Properties ◽  
Resting Membrane Potential ◽  
Hypothalamic Neurons ◽  
Spike Threshold ◽  
Spike Patterns

Spikes may play an important role in modulating a number of aspects of brain development. In early hypothalamic development, GABA can either evoke action potentials, or it can shunt other excitatory activity. In both slices and cultures of the mouse hypothalamus, we observed a heterogeneity of spike patterns and frequency in response to GABA. To examine the mechanisms underlying patterns and frequency of GABA-evoked spikes, we used conventional whole cell and gramicidin perforation recordings of neurons ( n = 282) in slices and cultures of developing mouse hypothalamus. Recorded with gramicidin pipettes, GABA application evoked action potentials in hypothalamic neurons in brain slices of postnatal day 2–9( P2- 9) mice. With conventional patch pipettes (containing 29 mM Cl−), action potentials were also elicited by GABA from neurons of 2–13 days in vitro (2–13 DIV) embryonic hypothalamic cultures. Depolarizing responses to GABA could be generally classified into three types: depolarization with no spike, a single spike, or complex patterns of multiple spikes. In parallel experiments in slices, electrical stimulation of GABAergic mediobasal hypothalamic neurons in the presence of glutamate receptor antagonists [10 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 100 μM 2-amino-5-phosphonopentanoic acid (AP5)] resulted in the occurrence of spikes that were blocked by bicuculline (20 μM). Blocking ionotropic glutamate receptors with AP5 and CNQX did not block GABA-mediated multiple spikes. Similarly, when synaptic transmission was blocked with Cd2+ (200 μM) and Ni2+(300 μM), GABA still induced multiple spikes, suggesting that the multiple spikes can be an intrinsic membrane property of GABA excitation and were not based on local interneurons. When the pipette [Cl−] was 29 or 45 mM, GABA evoked multiple spikes. In contrast, spikes were not detected with 2 or 10 mM intracellular [Cl−]. With gramicidin pipettes, we found that the mean reversal potential of GABA-evoked current ( E GABA) was positive to the resting membrane potential, suggesting a high intracellular [Cl−] in developing mouse neurons. Varying the holding potential from −80 to 0 mV revealed an inverted U-shaped effect on spike probability. Blocking voltage-dependent Na+ channels with tetrodotoxin eliminated GABA-evoked spikes, but not the GABA-evoked depolarization. Removing Ca2+ from the extracellular solution did not block spikes, indicating GABA-evoked Na+-based spikes. Although E GABA was more positive within 2–5 days in culture, the probability of GABA-evoked spikes was greater in 6- to 9-day cells. Mechanistically, this appears to be due to a greater Na+ current found in the older cells during a period when the E GABA is still positive to the resting membrane potential. GABA evoked similar spike patterns in HEPES and bicarbonate buffers, suggesting that Cl−, not bicarbonate, was primarily responsible for generatingmultiple spikes. GABA evoked either single or multiple spikes; neurons with multiple spikes had a greater Na+ current, a lower conductance, a more negative spike threshold, and a greater difference between the peak of depolarization and the spike threshold. Taken together, the present results indicate that the patterns of multiple action potentials evoked by GABA are an inherent property of the developing hypothalamic neuron.

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