scholarly journals Charybdotoxin blocks voltage-gated K+ channels in human and murine T lymphocytes.

1989 ◽  
Vol 93 (6) ◽  
pp. 1061-1074 ◽  
Author(s):  
S B Sands ◽  
R S Lewis ◽  
M D Cahalan
Keyword(s):  
T Lymphocytes ◽  
Time Course ◽  
Leukemia Cell ◽  
Lymphoid Cells ◽  
Jurkat Cells ◽  
Leukemia Cell Line ◽  
K Channel ◽  
K Channels ◽  
Voltage Gated ◽  
Scorpion Venoms

A variety of scorpion venoms and purified toxins were tested for effects on ion channels in human T lymphocytes, a human T leukemia cell line (Jurkat), and murine thymocytes, using the whole-cell patch-clamp method. Nanomolar concentrations of charbdotoxin (CTX), a purified peptide component of Leiurus quinquestriatus venom known to block Ca2+-activated K+ channels from muscle, blocked "type n" voltage-gated K+ channels in human T lymphoid cells. The Na+ channels occasionally expressed in these cells were unaffected by the toxin. From the time course of development and removal of K+ channel block we determined the rates of CTX binding and unbinding. CTX blocks K+ channels in Jurkat cells with a Kd value between 0.5 and 1.5 nM. Of the three types of voltage-gated K+ channels present in murine thymocytes, types n and n' are blocked by CTX at nanomolar concentrations. The third variety of K+ channels, "type l," is unaffected by CTX. Noxiustoxin (NTX), a purified toxin from Centruroides noxius known to block Ca2+-activated K+ channels, also blocked type n K+ channels with a high degree of potency (Kd = 0.2 nM). In addition, several types of crude scorpion venoms from the genera Androctonus, Buthus, Centruroides, and Pandinus blocked type n channels. We conclude that CTX and NTX are not specific for Ca2+ activated K+ channels and that purified scorpion toxins will provide useful probes of voltage-gated K+ channels in T lymphocytes. The existence of high-affinity sites for scorpion toxin binding may help to classify structurally related K+ channels and provide a useful tool for their biochemical purification.

scholarly journals Mitogen induction of ion channels in murine T lymphocytes.

1987 ◽  
Vol 89 (3) ◽  
pp. 405-420 ◽  
Author(s):  
T E Decoursey ◽  
K G Chandy ◽  
S Gupta ◽  
M D Cahalan
Keyword(s):  
T Lymphocytes ◽  
Time Course ◽  
K Channel ◽  
K Channels ◽  
Voltage Gated ◽  
Large Numbers ◽  
Predominant Type ◽  
Channel Expression ◽  
Activated T Lymphocytes ◽  
Cell Mitogen

Using gigohm-seal recording, we studied ion channel expression in resting and activated T lymphocytes from mice. Both the number of channels per cell and the predominant type of K+ channel depend upon the state of activation of the cell. Unstimulated T cells express small numbers of K+ channels, typically a dozen per cell, and are heterogeneous, usually expressing either type n or type l K+ channels (see DeCoursey, T. E., K. G. Chandy, S. Gupta, and M. D. Cahalan. 1987. Journal of General Physiology. 89:379-404). 1 d after stimulation by the murine T cell mitogen concanavalin A, large numbers of type n K+ channels appear in enlarged, activated cells. Type n channels appear in activated cells with a time course consistent with that reported for mitogen-induced enhancement of protein synthesis. Voltage-gated tetrodotoxin-sensitive Na+ channels present in about one-third of unstimulated cells from the MRL-n strain are increased approximately 10-fold after activation.

scholarly journals A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes

1998 ◽  
Vol 188 (9) ◽  
pp. 1593-1602 ◽  
Author(s):  
George R. Ehring ◽  
Hubert H. Kerschbaum ◽  
Claudia Eder ◽  
Amber L. Neben ◽  
Christopher M. Fanger ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Lines ◽  
K Channel ◽  
Na Channel ◽  
Activated T Cells ◽  
K Channels ◽  
Voltage Gated

The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin–resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl− channels. We propose that direct inhibition of K+ channels in T cells by progesterone contributes to progesterone-induced immunosuppression.

scholarly journals Non-Viral Transfection of Human T Lymphocytes

Processes ◽  
2018 ◽  
Vol 6 (10) ◽  
pp. 188 ◽  
Author(s):  
Simon Riedl ◽  
Patrick Kaiser ◽  
Alexander Raup ◽  
Christopher Synatschke ◽  
Valérie Jérôme ◽  
...  
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Mammalian Cells ◽  
Large Scale ◽  
Leukemia Cell ◽  
Jurkat Cells ◽  
Leukemia Cell Line ◽  
Human T Lymphocytes ◽  
Human T Cell ◽  
Viral Transfection

The genetic modification of human T lymphocytes with established non-viral methods is inefficient. Linear polyethylenimine (l-PEI), one of the most popular non-viral transfection agents for mammalian cells in general, only achieves transfection rates in the single digit percentage range for these cells. Here, a well-defined 24-armed poly(2-dimethylamino) ethyl methacrylate (PDMAEMA) nanostar (number average of the molecular weight: 755 kDa, polydispersity: <1.21) synthesized via atom transfer radical polymerization (ATRP) from a silsesquioxane initiator core is proposed as alternative. The agent is used to prepare polyplexes with plasmid DNA (pDNA). Under optimal conditions these polyplexes reproducibly transfect >80% of the cells from a human T-cell leukemia cell line (Jurkat cells) at viabilities close to 90%. The agent also promotes pDNA uptake when simply added to a mixture of cells and pDNA. This constitutes a particular promising approach for efficient transient transfection at large scale. Finally, preliminary experiments were carried out with primary T cells from two different donors. Results were again significantly better than for l-PEI, although further research into the response of individual T cells to the transfection agent will be necessary, before either method can be used to routinely transfect primary T lymphocytes.

scholarly journals Correlation between Charge Movement and Ionic Current during Slow Inactivation in Shaker K+ Channels

1997 ◽  
Vol 110 (5) ◽  
pp. 579-589 ◽  
Author(s):  
Riccardo Olcese ◽  
Ramón Latorre ◽  
Ligia Toro ◽  
Francisco Bezanilla ◽  
Enrico Stefani
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Ionic Current ◽  
K Channel ◽  
Charge Movement ◽  
Slow Inactivation ◽  
Gating Currents ◽  
Similar Time ◽  
K Channels ◽  
Recovery From Inactivation

Prolonged depolarization induces a slow inactivation process in some K+ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K+ channel and in the nonconducting mutant (Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K+ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.

scholarly journals Inactivation Gating of Kv4 Potassium Channels

1999 ◽  
Vol 113 (5) ◽  
pp. 641-660 ◽  
Author(s):  
Henry H. Jerng ◽  
Mohammad Shahidullah ◽  
Manuel Covarrubias
Keyword(s):  
Time Course ◽  
Membrane Potentials ◽  
Structural Basis ◽  
K Channel ◽  
Inactivation Curve ◽  
Double Mutation ◽  
K Channels ◽  
Closed State ◽  
Recovery From Inactivation ◽  
Inactivation Gating

Kv4 channels represent the main class of brain A-type K+ channels that operate in the subthreshold range of membrane potentials (Serodio, P., E. Vega-Saenz de Miera, and B. Rudy. 1996. J. Neurophysiol. 75:2174– 2179), and their function depends critically on inactivation gating. A previous study suggested that the cytoplasmic NH2- and COOH-terminal domains of Kv4.1 channels act in concert to determine the fast phase of the complex time course of macroscopic inactivation (Jerng, H.H., and M. Covarrubias. 1997. Biophys. J. 72:163–174). To investigate the structural basis of slow inactivation gating of these channels, we examined internal residues that may affect the mutually exclusive relationship between inactivation and closed-state blockade by 4-aminopyridine (4-AP) (Campbell, D.L., Y. Qu, R.L. Rasmussen, and H.C. Strauss. 1993. J. Gen. Physiol. 101:603–626; Shieh, C.-C., and G.E. Kirsch. 1994. Biophys. J. 67:2316–2325). A double mutation V[404,406]I in the distal section of the S6 region of the protein drastically slowed channel inactivation and deactivation, and significantly reduced the blockade by 4-AP. In addition, recovery from inactivation was slightly faster, but the pore properties were not significantly affected. Consistent with a more stable open state and disrupted closed state inactivation, V[404,406]I also caused hyperpolarizing and depolarizing shifts of the peak conductance–voltage curve (∼5 mV) and the prepulse inactivation curve (&gt;10 mV), respectively. By contrast, the analogous mutations (V[556,558]I) in a K+ channel that undergoes N- and C-type inactivation (Kv1.4) did not affect macroscopic inactivation but dramatically slowed deactivation and recovery from inactivation, and eliminated open-channel blockade by 4-AP. Mutation of a Kv4-specifc residue in the S4–S5 loop (C322S) of Kv4.1 also altered gating and 4-AP sensitivity in a manner that closely resembles the effects of V[404,406]I. However, this mutant did not exhibit disrupted closed state inactivation. A kinetic model that assumes coupling between channel closing and inactivation at depolarized membrane potentials accounts for the results. We propose that components of the pore's internal vestibule control both closing and inactivation in Kv4 K+ channels.

scholarly journals Potassium channel block by internal calcium and strontium.

1991 ◽  
Vol 97 (3) ◽  
pp. 627-638 ◽  
Author(s):  
C M Armstrong ◽  
Y Palti
Keyword(s):  
Time Course ◽  
External Solution ◽  
High Energy ◽  
K Channel ◽  
Giant Fiber ◽  
Distance Measurements ◽  
K Channels ◽  
High Energy Barrier ◽  
Intracellular Ca ◽  
A Site

We show that intracellular Ca blocks current flow through open K channels in squid giant fiber lobe neurons. The block has similarities to internal Sr block of K channels in squid axons, which we have reexamined. Both ions must cross a high energy barrier to enter the blocking site from the inside, and block occurs only with millimolar concentrations and with strong depolarization. With Sr (axon) or Ca (neuron) inside, IK is normal in time course for voltages less than about +50 mV; but for large steps, above +90 mV, there is a rapid time-dependent block or "inactivation." From roughly +70 to +90 mV (depending on concentration) the current has a complex time course that may be related to K accumulation near the membrane's outer surface. Block can be deepened by either increasing the concentration or the voltage. Electrical distance measurements suggest that the blocking ion moves to a site deep in the channel, possibly near the outer end. Block by internal Ca can be prevented by putting 10 mM Rb in the external solution. Recovery from block after a strong depolarization occurs quickly at +30 mV, with a time course that is about the same as that of normal K channel activation at this voltage. 20 mM Mg in neurons had no discernible blocking effect. The experiments raise questions regarding the relation of block to normal channel gating. It is speculated that when the channel is normally closed, the "blocking" site is occupied by a Ca ion that comes from the external medium.

scholarly journals Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen.

1992 ◽  
Vol 100 (3) ◽  
pp. 401-426 ◽  
Author(s):  
M D Ganfornina ◽  
J López-Barneo
Keyword(s):  
Time Course ◽  
Single Channel ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Glomus Cells ◽  
Control Value ◽  
Voltage Dependent ◽  
K Current ◽  
Chemoreceptor Cells

Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.

scholarly journals Gating of Recombinant Small-Conductance Ca-activated K+ Channels by Calcium

1998 ◽  
Vol 111 (4) ◽  
pp. 565-581 ◽  
Author(s):  
Birgit Hirschberg ◽  
James Maylie ◽  
John P. Adelman ◽  
Neil V. Marrion
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Cell Types ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Sensitive Clone ◽  
Single Channel Currents ◽  
Open States ◽  
Small Conductance

Small-conductance Ca-activated K+ channels play an important role in modulating excitability in many cell types. These channels are activated by submicromolar concentrations of intracellular Ca2+, but little is known about the gating kinetics upon activation by Ca2+. In this study, single channel currents were recorded from Xenopus oocytes expressing the apamin-sensitive clone rSK2. Channel activity was detectable in 0.2 μM Ca2+ and was maximal above 2 μM Ca2+. Analysis of stationary currents revealed two open times and three closed times, with only the longest closed time being Ca dependent, decreasing with increasing Ca2+ concentrations. In addition, elevated Ca2+ concentrations resulted in a larger percentage of long openings and short closures. Membrane voltage did not have significant effects on either open or closed times. The open probability was ∼0.6 in 1 μM free Ca2+. A lower open probability of ∼0.05 in 1 μM Ca2+ was also observed, and channels switched spontaneously between behaviors. The occurrence of these switches and the amount of time channels spent displaying high open probability behavior was Ca2+ dependent. The two behaviors shared many features including the open times and the short and intermediate closed times, but the low open probability behavior was characterized by a different, long Ca2+-dependent closed time in the range of hundreds of milliseconds to seconds. Small-conductance Ca- activated K+ channel gating was modeled by a gating scheme consisting of four closed and two open states. This model yielded a close representation of the single channel data and predicted a macroscopic activation time course similar to that observed upon fast application of Ca2+ to excised inside-out patches.

PI3K/AKT Pathway as a Potential Therapeutic Target In Myelodysplastic Syndrome

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1871-1871
Author(s):  
Denise C Rezende ◽  
Lorena Zaida Pacheco ◽  
Luis Arthur F. Pelloso ◽  
Maria L. Chauffaille ◽  
Marçal C.A Silva ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Leukemia Cell ◽  
Jurkat Cells ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Akt Pathway ◽  
Akt Activation ◽  
Cd34 Cells

Abstract Abstract 1871 Introduction: PI3K/AKT pathway is involved in cell growth, proliferation and apoptosis. A key downstream effector is the phosphorylated serine-threonine Akt (p-AKT). Constitutive activation of PI3K/AKT has been observed in solid tumours and leukemic cells. Inhibition of PI3K/AKT activity, results in apoptosis in cell lines (CL) after treatment with different compounds, e.g. deguelin, a natural product from the leguminous Mundulea sericea, with antitumour effects. Aims: To evaluate PI3K/AKT activation in MDS patients and its therapeutic potential in MDS. Methods: PI3K/AKT activation was evaluated by flow cytometry (FC) using an alexa-fluor 488-antibody Ser 473 p-AKT (Cell Signalling Technology). A triple immunostaining procedure using CD45-PerCP and CD34-PE was used for p-AKT expression in CD34+ primary samples. The p-AKT activity was determined using Kolmogorov-Smirnov test (D). CD34+ cells from healthy donors and Jurkat cells were used as negative and positive controls respectively. Apoptosis (determined by Annexin V and PI/7AAD) and cell cycle arrest (using RNAse and PI) were determined following treatments with LY294002 (50uM), and deguelin (100-500nM) in P-39 myeloid leukemia cell line, with constitutive PI3K/AKT activation. Apoptosis was determined in bone marrow mononuclear cells and CD34+ cells from MDS patients with the same treatments. To evaluate in vivo activity of deguelin, we used a xenotransplant model. Briefly, NODSCID mice were injected intrafemurally with P-39 CL and 12 days post transplant a three week-course of treatment, every other day, was started (deguelin 4mg/Kg, n=3 vs vehicle, n=3). Results: P-39 CL showed constitutive PI3K/AKT activation with levels significantly higher than in CD34+cells from controls (median±SD= 0.73. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Changes in the expression of potassium channels during mouse T cell development.

1986 ◽  
Vol 164 (6) ◽  
pp. 1846-1861 ◽  
Author(s):  
D McKinnon ◽  
R Ceredig
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Cell Lineage ◽  
Electrophysiological Recording ◽  
K Channel ◽  
Delayed Rectifier ◽  
Developmentally Regulated ◽  
K Channels ◽  
Channel Expression ◽  
Low Levels

In this report we have combined the whole-cell electrophysiological recording technique with flow microfluorometry to isolate phenotypically defined thymocytes and T lymphocytes. Results obtained showed that J11d-/Lyt-2-/L3T4- cells express none or very few delayed rectifier K+ channels, whereas most other Lyt-2-/L3T4- cells, as well as typical cortical thymocytes (Lyt-2+/L3T4+), do express K+ channels. Mature (Lyt-2+/L3T4- or Lyt-2-/L3T4+) thymocytes, which are heterogeneous for J11d expression, were also found to be heterogeneous for K+ channel expression. Consistent with this finding was the observation that the cortisone-resistant subpopulation of thymocytes, which express low levels of J11d, were enriched for cells expressing low levels of K+ channels. Mature phenotype peripheral T lymphocytes expressed very low levels of K+ channels, but upon activation with Con A were found to express high levels of K+ channels. The results suggest that K+ channel expression in T cells is developmentally regulated. Increased expression of the channel is induced in response to mitogenic signals throughout the T cell lineage. Expression of the channel, therefore, serves as a useful marker in defining steps in the T cell differentiation pathway.

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