scholarly journals Calcium-activated potassium channels in resting and activated human T lymphocytes. Expression levels, calcium dependence, ion selectivity, and pharmacology.

1993 ◽  
Vol 102 (4) ◽  
pp. 601-630 ◽  
Author(s):  
S Grissmer ◽  
A N Nguyen ◽  
M D Cahalan
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Ion Selectivity ◽  
Hill Coefficient ◽  
Ca Channel ◽  
Ca Channels ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
Whole Cell ◽  
Unitary Conductance

Ca(2+)-activated K+[K(Ca)] channels in resting and activated human peripheral blood T lymphocytes were characterized using simultaneous patch-clamp recording and fura-2 monitoring of cytosolic Ca2+ concentration, [Ca2+]i. Whole-cell experiments, using EGTA-buffered pipette solutions to raise [Ca2+]i to 1 microM, revealed a 25-fold increase in the number of conducting K(Ca) channels per cell, from an average of 20 in resting T cells to > 500 channels per cell in T cell blasts after mitogenic activation. The opening of K(Ca) channels in both whole-cell and inside-out patch experiments was highly sensitive to [Ca2+]i (Hill coefficient of 4, with a midpoint of approximately 300 nM). At optimal [Ca2+]i, the open probability of a K(Ca) channel was 0.3-0.5. K(Ca) channels showed little or no voltage dependence from -100 to 0 mV. Single-channel I-V curves were linear with a unitary conductance of 11 pS in normal Ringer and exhibited modest inward rectification with a unitary conductance of approximately 35 pS in symmetrical 160 mM K+. Permeability ratios, relative to K+, determined from reversal potential measurements were: K+ (1.0) > Rb+ (0.96) > NH4+ (0.17) > Cs+ (0.07). Slope conductance ratios were: NH4+ (1.2) > K+ (1.0) > Rb+ (0.6) > Cs+ (0.10). Extracellular Cs+ or Ba2+ each induced voltage-dependent block of K(Ca) channels, with block increasing at hyperpolarizing potentials in a manner suggesting a site of block 75% across the membrane field from the outside. K(Ca) channels were blocked by tetraethylammonium (TEA) applied externally (Kd = 40 mM), but were unaffected by 10 mM TEA applied inside by pipette perfusion. K(Ca) channels were blocked by charybdotoxin (CTX) with a half-blocking dose of 3-4 nM, but were resistant to block by noxiustoxin (NTX) at 1-100 nM. Unlike K(Ca) channels in Jurkat T cells, the K(Ca) channels of normal resting or activated T cells were not blocked by apamin. We conclude that while K(Ca) and voltage-gated K+ channels in the same cells share similarities in ion permeation, Cs+ and Ba2+ block, and sensitivity to CTX, the underlying proteins differ in structural characteristics that determine channel gating and block by NTX and TEA.

Differential responses of Ca-activated K channels to bradykinin in sensory neurons and F-11 cells

1992 ◽  
Vol 262 (2) ◽  
pp. C453-C460 ◽  
Author(s):  
K. Naruse ◽  
D. S. McGehee ◽  
G. S. Oxford
Keyword(s):  
Intracellular Signaling ◽  
Single Channel ◽  
Outward Current ◽  
Transient Outward Current ◽  
Ca Channel ◽  
Ca Channels ◽  
Drg Neurons ◽  
Open Probability ◽  
K Channels ◽  
Drg Neuron

The nonapeptide bradykinin (BK) excites a subset of dorsal root ganglion (DRG) neurons with putative nociceptive functions by stimulating an inward cation current. In addition, BK stimulates various intracellular signaling pathways including an elevation of intracellular Ca2+. In a DRG neuron x neuroblastoma hybrid cell (F-11), BK stimulates similar increases in intracellular [Ca2+] and inward current but also elicits a large transient outward current through Ca(2+)-activated K channels. We have investigated the mechanisms underlying differential expression of outward current responses in the two cell types at the single channel level. Although K(Ca) channel activity appears in inside-out patches from both cells exposed to Ca2+, BK applied to the extrapatch membrane of cell-attached patches activates K(Ca) channels in F-11 but not DRG neurons. Whereas single K(Ca) channels are quantitatively similar in terms of conductance, voltage-dependence, and sensitivity to tetraethylammonium, they differ in sensitivity to intracellular Ca2+. Channel activation in both cells requires at least four Ca2+ ions, but half-maximal activation occurs at slightly higher [Ca2+] for DRG neurons. The shift in the Ca2+ dose-response curve combined with the steep [Ca2+] dependence of channel open probability makes it less likely that a BK-induced rise in internal [Ca2+] induced will trigger a transient outward current and resultant hyperpolarization in a DRG neuron.

scholarly journals Angiotensin II stimulates epithelial sodium channels in the cortical collecting duct of the rat kidney

2012 ◽  
Vol 302 (6) ◽  
pp. F679-F687 ◽  
Author(s):  
Peng Sun ◽  
Peng Yue ◽  
Wen-Hui Wang
Keyword(s):  
Angiotensin Ii ◽  
Single Channel ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Ang Ii ◽  
Cortical Collecting Duct ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
Whole Cell ◽  
Na Currents

We examined the effect of angiotensin II (ANG II) on epithelial Na+channel (ENaC) in the rat cortical collecting duct (CCD) with single-channel and the perforated whole cell patch-clamp recording. Application of 50 nM ANG II increased ENaC activity, defined by NPo(a product of channel numbers and open probability), and the amiloride-sensitive whole cell Na currents by twofold. The stimulatory effect of ANG II on ENaC was absent in the presence of losartan, suggesting that the effect of ANG II on ENaC was mediated by ANG II type 1 receptor. Moreover, depletion of intracellular Ca2+with 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA)-AM failed to abolish the stimulatory effect of ANG II on ENaC but inhibiting protein kinase C (PKC) abolished the effect of ANG II, suggesting that the effect of ANG II was the result of stimulating Ca2+-independent PKC. This notion was also suggested by the experiments in which stimulation of PKC with phorbol ester derivative mimicked the effect of ANG II and increased amiloride-sensitive Na currents in the principal cell, an effect that was not abolished by treatment of the CCD with BAPTA-AM. Also, inhibition of NADPH oxidase (NOX) with diphenyleneiodonium chloride abolished the stimulatory effect of ANG II on ENaC and application of superoxide donors, pyrogallol or xanthine and xanthine oxidase, significantly increased ENaC activity. Moreover, addition of ANG II or H2O2diminished the arachidonic acid (AA)-induced inhibition of ENaC in the CCD. We conclude that ANG II stimulates ENaC in the CCD through a Ca2+-independent PKC pathway that activates NOX thereby increasing superoxide generation. The stimulatory effect of ANG II on ENaC may be partially the result of blocking AA-induced inhibition of ENaC.

Prepulse-induced mode 2 gating behavior with and without β-adrenergic stimulation in cardiac L-type Ca channels

1999 ◽  
Vol 276 (6) ◽  
pp. C1338-C1345 ◽  
Author(s):  
Yuji Hirano ◽  
Takashi Yoshinaga ◽  
Mitsushige Murata ◽  
Masayasu Hiraoka
Keyword(s):  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Ca Channel ◽  
Ca Channels ◽  
Adrenergic Stimulation ◽  
Open Probability ◽  
Mode 2 ◽  
Voltage Dependent ◽  
Phosphorylation Mechanism ◽  
Prepulse Facilitation

Mode 2 gating of L-type Ca channels is characterized by high channel open probability ( NP o) and long openings. In cardiac myocytes, this mode is evoked physiologically in two apparently different circumstances: membrane depolarization (prepulse facilitation) and activation of protein kinase A. To examine whether the phosphorylation mechanism is involved during prepulse-induced facilitation of cardiac L-type Ca channels, we used isolated guinea pig ventricular myocytes to analyze depolarization-induced modal gating behavior under different basal levels of phosphorylation. In control, NP o measured at 0 mV was augmented as the duration of prepulse to +100 mV was prolonged from 50 to 400 ms. This was due to the induction of mode 2 gating behavior clustered at the beginning of test pulses. Analysis of open time distribution revealed that the prepulse evoked an extra component, the time constant of which is not dependent on prepulse duration. When isoproterenol (1 μM) was applied to keep Ca channels at an enhanced level of phosphorylation, basal NP o without prepulse was increased by a factor of 3.6 ± 2.2 ( n = 6). Under these conditions, prepulse further increased NP o by promoting long openings with the same kinetics of transition to mode 2 gating (τ ≅ 200 ms at +100 mV). Likewise, recovery from mode 2 gating, as estimated by the decay of averaged unitary current, was not affected after β-stimulation (τ ≅ 25 ms at 0 mV). The kinetic behavior independent from the basal level of phosphorylation or activity of cAMP-dependent protein kinase suggests that prepulse facilitation of the cardiac Ca channel involves a mechanism directly related to voltage-dependent conformational change rather than voltage-dependent phosphorylation.

Abstract 174: Biophysical Basis of Protein Kinase C Inhibition of the Novel alpha1D Calcium Channel

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Mohamed Chahine ◽  
Yongxia Qu ◽  
Mohamed Boutjdir
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Calcium Channel ◽  
Single Channel ◽  
Ca Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Whole Cell ◽  
Kinase C ◽  
Pkc Activation

The recently reported α 1D calcium channel in the heart is known to be regulated by protein kinase C (PKC) at the whole cell level and has been implicated in atrial fibrillation. The biophysical basis of this regulation at the single channel level is not known. Therefore, the effect of PKC activation was studied on α 1D calcium channel expressed in tsA201 cells using cell-attached method. Unitary currents were recorded in the presence of 70 mM Ba 2+ as the charge carrier. Unitary currents were evoked by 500 ms depolarizing pulses from a holding potential of −80 mV every 0.5 Hz. Under basal condition, channel activity was rare and infrequent, however Bay K 8644 (1 μM) induced channel openings with a conductance of 22.3 pS. Single channel analysis of open and closed time distributions were best fitted with a single exponential. PKC activation by PMA (10 nM), a phorbol ester derivative, resulted in a decrease in open probability and increase in closed-time without any significant effect on the conductance of the α 1D calcium channel. This is consistent with a decreased entry of α 1D Ca channel into open states in the presence of PMA. These data show, for the fist time, 1) the α 1D calcium channel activity at the single channel level and 2) the biophysical basis of by which PKC activation inhibits the α 1D calcium channel. The shortening of the open-time and the lengthening of the closed-time constants and the increase in blank sweeps may explain the inhibition of the α 1D Ca-channel activity and the reduction in whole-cell α 1D Ca current previously reported. Altogether, these data are relevant to the understanding of the patho-physiology of α 1D calcium channel and its regulation by the autonomics.

Regulation of cardiac L-type Ca channels in planar lipid bilayers by G proteins and protein phosphorylation

1993 ◽  
Vol 264 (6) ◽  
pp. C1473-C1479 ◽  
Author(s):  
Y. Wang ◽  
C. Townsend ◽  
R. L. Rosenberg
Keyword(s):  
G Proteins ◽  
Lipid Bilayers ◽  
Okadaic Acid ◽  
Initial Level ◽  
Planar Lipid Bilayers ◽  
Ca Channel ◽  
Ca Channels ◽  
Channel Activity ◽  
Open Probability ◽  
Gs Alpha

We have studied the effects of activated G proteins (Gs alpha and Gi1 alpha), adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA), and okadaic acid on L-type Ca channels incorporated from porcine ventricular sarcolemma into planar lipid bilayers. Channel activity evoked by membrane depolarizations diminished to extremely low levels within 2 min of incorporation (channel "rundown"). When Gs alpha [activated with guanosine 5'-O-(3-thiotriphosphate)] was present in the intracellular chamber, the initial level of channel activity was increased and rundown was delayed, so that channel activity was sustained for longer times after incorporation. The effect was specific for activated Gs alpha; activated Gi1 alpha, heat-denatured, activated Gs alpha, and unactivated Gs alpha did not augment channel activity. Activated Gi1 alpha inhibited the stimulation of Ca channel activity by Gs alpha. Treatment of the sarcolemmal membranes with PKA and Mg-ATP also increased the initial channel open probability and delayed their rundown. Addition of intracellular Gs alpha to PKA-treated channels increased the initial level of activity above that seen with PKA or Gs alpha alone, suggesting different nonocclusive pathways for the channel stimulation. This was also supported by the observation that activated Gi1 alpha had no effect on PKA-treated channels. Okadaic acid (100 nM) increased the level of Ca channel activity, suggesting that dephosphorylation by endogenous phosphatases participated in the downregulation of the channels in cell-free membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.

1989 ◽  
Vol 1 (1) ◽  
pp. 99-112 ◽  
Author(s):  
R S Lewis ◽  
M D Cahalan
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
T Cell Activation ◽  
Cell Activation ◽  
Free Calcium ◽  
Patch Clamp Recording ◽  
Activated T Cells ◽  
Whole Cell ◽  
Fura 2 ◽  
Perforated Patch

A rapid rise in the level of cytosolic free calcium ([Ca2+]i) is believed to be one of several early triggering signals in the activation of T lymphocytes by antigen. Although Ca2+ release from intracellular stores and its contribution to Ca2+ signaling in many cell types is well documented, relatively little is known regarding the role and mechanism of Ca2+ entry across the plasma membrane. We have investigated mitogen-triggered Ca2+ signaling in individual cells of the human T-leukemia-derived line, Jurkat, using fura-2 imaging and patch-clamp recording techniques. Phytohemagglutinin (PHA), a mitogenic lectin, induces repetitive [Ca2+]i oscillations in these cells peaking at micromolar levels with a period of 90-120 s. The oscillations depend critically upon Ca2+ influx across the plasma membrane, as they are rapidly terminated by removal of extracellular Ca2+, addition of Ca(2+)-channel blockers such as Ni2+ or Cd2+, or membrane depolarization. Whole-cell and perforated-patch recording methods were combined with fura-2 measurements to identify the mitogen-activated Ca2+ conductance involved in this response. A small, highly selective Ca2+ conductance becomes activated spontaneously in whole-cell recordings and in response to PHA in perforated-patch experiments. This conductance has properties consistent with a role in T-cell activation, including activation by PHA, lack of voltage-dependent gating, inhibition by Ni2+ or Cd2+, and regulation by intracellular Ca2+. Moreover, a tight temporal correlation between oscillations of Ca2+ conductance and [Ca2+]i suggests a role for the membrane Ca2+ conductance in generating [Ca2+]i oscillations in activated T cells.

scholarly journals A population density and moment-based approach to modeling domain Ca-mediated inactivation of L-type Ca channels

2015 ◽  
Author(s):  
Xiao Wang ◽  
Kiah Hardcastle ◽  
Seth H Weinberg ◽  
Gregory D Smith
Keyword(s):  
Population Density ◽  
Time Constant ◽  
Traditional Approach ◽  
Markov Chain Model ◽  
Order Moment ◽  
Chain Model ◽  
Ca Channel ◽  
Ca Channels ◽  
Moment Equations ◽  
Whole Cell

We present a population density and moment-based description of the stochastic dynamics of domain Ca-mediated inactivation of L-type Ca channels. Our approach accounts for the effect of heterogeneity of local Ca signals on whole cell Ca currents; however, in contrast with prior work, e.g., Sherman et al. (1990), we do not assume that Ca domain formation and collapse are fast compared to channel gating. We demonstrate the population density and moment-based modeling approaches using a 12-state Markov chain model of an L-type Ca channel introduced by Greenstein and Winslow (2002). Simulated whole cell voltage clamp responses yield an inactivation function for the whole cell Ca current that agrees with the traditional approach when domain dynamics are fast. We analyze the voltage-dependence of Ca inactivation that may occur via slow heterogeneous domains. Next, we find that when channel permeability is held constant, Ca-mediated inactivation of L-type channel increases as the domain time constant increases, because a slow domain collapse rate leads to increased mean domain [Ca] near open channels; conversely, when the maximum domain [Ca] is held constant, inactivation decreases as the domain time constant increases. Comparison of simulation results using population densities and moment equations confirms the computational efficiency of the moment-based approach, and enables the validation of two distinct methods of truncating and closing the open system of moment equations. In general, a slow domain time constant requires higher order moment truncation for agreement between moment-based and population density simulations.

Properties of ATP-gated channels recorded from rat sympathetic neurons: voltage dependence and regulation by Zn2+ ions

1995 ◽  
Vol 73 (1) ◽  
pp. 312-319 ◽  
Author(s):  
R. Cloues
Keyword(s):  
Voltage Dependence ◽  
Sympathetic Neurons ◽  
Receptor Activation ◽  
P2x Receptor ◽  
Allosteric Modulator ◽  
Ion Permeation ◽  
Open Probability ◽  
Whole Cell ◽  
Unitary Conductance ◽  
Low Concentrations

1. I recorded ATP-gated channels from excised outside-out patches from rat sympathetic neurons. The channels had a unitary conductance of approximately 12 pS at -80 mV, were activated only when ATP was present in the bath, and could be blocked by the P2-purinoceptor antagonist suramin. 2. The open probability of the channels showed a strong voltage dependence, increasing significantly with hyperpolarization. This suggests that whole cell rectification of currents evoked by P2X receptor activation is a result of channel voltage dependence and not of properties of ion permeation. 3. Low concentrations of extracellular Zn2+, which has previously been shown to potentiate whole cell ATP-evoked currents, increased the open probability of ATP-gated channels without altering the unitary conductance. 4. Zn2+ increased both the opening frequency and the burst duration of ATP-gated channels. The results are consistent with Zn2+ acting as an allosteric modulator of the P2X receptor in sympathetic neurons.

scholarly journals Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells.

1986 ◽  
Vol 88 (3) ◽  
pp. 293-319 ◽  
Author(s):  
P Hess ◽  
J B Lansman ◽  
R W Tsien
Keyword(s):  
Binding Sites ◽  
Single Channel ◽  
Heart Cells ◽  
Ca Channel ◽  
Monovalent Cations ◽  
Ca Channels ◽  
Whole Cell ◽  
Double Occupancy ◽  
Monovalent Ions ◽  
Ca Ions

Single channel and whole cell recordings were used to study ion permeation through Ca channels in isolated ventricular heart cells of guinea pigs. We evaluated the permeability to various divalent and monovalent cations in two ways, by measuring either unitary current amplitude or reversal potential (Erev). According to whole cell measurements of Erev, the relative permeability sequence is Ca2+ greater than Sr2+ greater than Ba2+ for divalent ions; Mg2+ is not measurably permeant. Monovalent ions follow the sequence Li+ greater than Na+ greater than K+ greater than Cs+, and are much less permeant than the divalents. These whole cell measurements were supported by single channel recordings, which showed clear outward currents through single Ca channels at strong depolarizations, similar values of Erev, and similar inflections in the current-voltage relation near Erev. Information from Erev measurements stands in contrast to estimates of open channel flux or single channel conductance, which give the sequence Na+ (85 pS) greater than Li+ (45 pS) greater than Ba2+ (20 pS) greater than Ca2+ (9 pS) near 0 mV with 110-150 mM charge carrier. Thus, ions with a higher permeability, judged by Erev, have lower ion transfer rates. In another comparison, whole cell Na currents through Ca channels are halved by less than 2 microM [Ca]o, but greater than 10 mM [Ca]o is required to produce half-maximal unitary Ca current. All of these observations seem consistent with a recent hypothesis for the mechanism of Ca channel permeation, which proposes that: ions pass through the pore in single file, interacting with multiple binding sites along the way; selectivity is largely determined by ion affinity to the binding sites rather than by exclusion by a selectivity filter; occupancy by only one Ca ion is sufficient to block the pore's high conductance for monovalent ions like Na+; rapid permeation by Ca ions depends upon double occupancy, which only becomes significant at millimolar [Ca]o, because of electrostatic repulsion or some other interaction between ions; and once double occupancy occurs, the ion-ion interaction helps promote a quick exit of Ca ions from the pore into the cell.

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