The ACh-induced whole-cell currents in sheep parotid secretory cells. Do BK channels really carry the ACh-evoked whole-cell K+ current?

A number of Drosophila learning mutants have defective intracellular second-messenger systems. In an effort to develop techniques that will allow direct measurement of the effects of these mutations on whole-cell neuronal membrane currents, the perforated-patch whole-cell (PPWC) technique has been applied to cleavage-arrested cultured embryonic Drosophila neurons. This technique permits the measurement of membrane currents without disturbing the intracellular environment. As a result of the maintenance of the intracellular environment, Drosophila neuron currents are found to be much more stable than when measured using the conventional whole-cell (CWC) patch-clamp technique. Ca2+ channel currents, which typically 'wash out' within a few minutes of the beginning of CWC recording, are stable for the duration of the seal (tens of minutes) when measured using the PPWC technique. Since the learning mutations dunce and rutabaga disrupt cyclic AMP signalling, the action of externally applied dibutyryl cyclic AMP (db-cAMP) and theophylline on Ca2+ and K+ channel currents were studied. db-cAMP and theophylline enhanced the Ba2+ current, carried by Ca2+ channels, but had no effect on the K+ current in the cleavage-arrested neurons. However, the large variability and reduction in density of Ba2+ and K+ currents raise questions about the suitability of using these cleavage-arrested cells as models for Drosophila neurons.

Download Full-text

Guinea pig visceral C-fiber neurons are diverse with respect to the K+ currents involved in action-potential repolarization

Journal of Neurophysiology ◽

10.1152/jn.1994.71.2.561 ◽

1994 ◽

Vol 71 (2) ◽

pp. 561-574 ◽

Cited By ~ 16

Author(s):

E. P. Christian ◽

J. Togo ◽

K. E. Naper

Keyword(s):

Guinea Pig ◽

Action Potential ◽

Outward Current ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Whole Cell ◽

C Fiber ◽

K Current ◽

Action Potential Repolarization

1. Intracellular recordings were made from C-fiber neurons identified by antidromic conduction velocity in intact guinea pig nodose ganglia maintained in vitro, and whole-cell patch clamp recordings were made from dissociated guinea pig nodose neurons to investigate the contribution of various K+ conductances to action-potential repolarization. 2. The repolarizing phase of the intracellularly recorded action potential was prolonged in a concentration-dependent manner by charybdotoxin (Chtx; EC50 = 39 nM) or iberiatoxin (Ibtx; EC50 = 48 nM) in a subpopulation of 16/36 C-fiber neurons. In a subset of these experiments, removal of extracellular Ca2+ reversibly prolonged action-potential duration (APD) in the same 4/9 intracellularly recorded C-fiber neurons affected by Chtx (> or = 100 nM). These convergent results support that a Ca(2+)-activated K+ current (IC) contributes to action-potential repolarization in a restricted subpopulation of C-fiber neurons. 3. Tetraethylammonium (TEA; 1-10 mM) increased APD considerably further in the presence of 100-250 nM Chtx or Ibtx, or in nominally Ca(2+)-free superfusate in 14/14 intracellularly recorded C-fiber neurons. TEA affected APD similarly in subpopulations of neurons with and without IC, suggesting that a voltage-dependent K+ current (IK) contributes significantly to action-potential repolarization in most nodose C-fiber neurons. 4. Substitution of Mn2+ for Ca2+ reduced outward whole-cell currents elicited by voltage command steps positive to -30 mV (2-25 ms) in a subpopulation of 21/36 dissociated nodose neurons, supporting the heterogeneous expression of IC. The kinetics of outward tail current relaxations (tau s of 1.5-2 ms) measured at the return of 2-3 ms depolarizing steps to -40 mV were indistinguishable in neurons with and without IC, precluding a separation of the nodose IC and IK by a difference in deactivation rates. 5. Chtx (10-250 nM) reduced in a subpopulation of 3/8 C-fiber neurons the total outward current elicited by voltage steps depolarized to -30 mV in single microelectrode voltage-clamp recordings. TEA (5-10 mM) further reduced outward current in the presence of 100-250 nM Chtx in all eight experiments. The Chtx-sensitive current was taken to represent IC, and the TEA-sensitive current, the IK component contributing to action-potential repolarization. 6. Rapidly inactivating current (IA) was implicated in action-potential repolarization in a subpopulation of intracellularly recorded C-fiber neurons. In 4/7 neurons, incremented hyperpolarizing prepulses negative to -50 mV progressively shortened APD.(ABSTRACT TRUNCATED AT 400 WORDS)

Download Full-text

BK Channels in Human Glioma Cells

Journal of Neurophysiology ◽

10.1152/jn.2001.85.2.790 ◽

2001 ◽

Vol 85 (2) ◽

pp. 790-803 ◽

Cited By ~ 83

Author(s):

Christopher B. Ransom ◽

Harald Sontheimer

Keyword(s):

Rank Order ◽

Glioma Cells ◽

Cell Types ◽

Bk Channels ◽

Human Glioma ◽

Human Glioma Cells ◽

Whole Cell ◽

Channel Expression ◽

Intracellular Ca ◽

Current Activation

Ion channels in inexcitable cells are involved in proliferation and volume regulation. Glioma cells robustly proliferate and undergo shape and volume changes during invasive migration. We investigated ion channel expression in two human glioma cell lines (D54MG and STTG-1). With low [Ca2+]i, both cell types displayed voltage-dependent currents that activated at positive voltages (more than +50 mV). Current density was sensitive to intracellular cation replacement with the following rank order; K+ > Cs+ ≈ Li+ > Na+. Currents were >80% inhibited by iberiotoxin (33 nM), charybdotoxin (50 nM), quinine (1 mM), tetrandrine (30 μM), and tetraethylammonium ion (TEA; 1 mM). Extracellular phloretin (100 μM), an activator of BK(Ca2+) channels, and elevated intracellular Ca2+ negatively shifted the I-V curve of whole cell currents. With 0, 0.1, and 1 μM [Ca2+]i, the half-maximal voltages, V 0.5, for whole cell current activation were +150, +65, and +12 mV, respectively. Elevating [K+]o potentiated whole cell currents in a fashion proportional to the square-root of [K+]o. Recording from cell-attached patches revealed large conductance channels (150–200 pS) with similar voltage dependence and activation kinetics as whole cell currents. These data indicate that human glioma cells express large-conductance, Ca2+-activated K+ (BK) channels. In amphotericin-perforated patches bradykinin (1 μM) activated TEA-sensitive currents that were abolished by preincubation with bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid-AM (BAPTA-AM). The BK channels described here may influence the responses of glioma cells to stimuli that increase [Ca2+]i.

Download Full-text

Opening of large-conductance calcium-activated potassium channels by the substituted benzimidazolone NS004

Journal of Neurophysiology ◽

10.1152/jn.1994.71.5.1873 ◽

1994 ◽

Vol 71 (5) ◽

pp. 1873-1882 ◽

Cited By ~ 46

Author(s):

M. C. McKay ◽

S. I. Dworetzky ◽

N. A. Meanwell ◽

S. P. Olesen ◽

P. H. Reinhart ◽

...

Keyword(s):

Rat Brain ◽

Lipid Bilayers ◽

Single Channel ◽

Outward Current ◽

Calcium Sensitivity ◽

Bk Channels ◽

Gh3 Cells ◽

Xenopus Laevis Oocytes ◽

Planar Lipid Bilayers ◽

Whole Cell

1. We used electrophysiological techniques to examine the effects of 5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidaz ole- 2-one (NS004) on large-conductance calcium-activated potassium (BK) channels. 2. We used recordings from excised membrane patches (cell-attached and inside-out single-channel configurations) and whole-cell patch-clamp recordings to examine the effects of NS004 on single BK channels and whole-cell outward currents, respectively, in rat GH3 clonal pituitary tumor cells. We also tested NS004 on voltage-clamped BK channels isolated from rat brain plasma membrane preparations and reconstituted into planar lipid bilayers. Finally, we used two-electrode voltage-clamp techniques to study the effects of NS004 on currents expressed in Xenopus laevis oocytes by the recently described Slo BK clone from Drosophila. 3. In GH3 cells and in Xenopus oocytes expressing the Slo gene product NS004 produced an increase in an iberiotoxin- or tetraethylammonium-sensitive whole-cell outward current, respectively. NS004 produced a significant increase in the activity of single GH3 cell BK channels and rat brain BK channels reconstituted into planar lipid bilayers. In both systems this was characterized by an increase in channel mean open time, a decrease in interburst interval, and an apparent increase in channel voltage/calcium sensitivity. 4. These data indicate that NS004 could be useful for investigating the biophysical and molecular properties of BK channels and for determining the functional consequences of the opening of BK channels.

Download Full-text

Whole-cell K+ current across the plasma membrane of guard cells from a grass: Zea mays

Planta ◽

10.1007/bf00196258 ◽

1992 ◽

Vol 186 (2) ◽

Cited By ~ 25

Author(s):

K.A. Fairley-Grenot ◽

S.M. Assmann

Keyword(s):

Zea Mays ◽

Plasma Membrane ◽

Guard Cells ◽

Whole Cell ◽

K Current

Download Full-text

Large-conductance calcium-activated potassium current modulates excitability in isolated canine intracardiac neurons

AJP Cell Physiology ◽

10.1152/ajpcell.00148.2012 ◽

2013 ◽

Vol 304 (3) ◽

pp. C280-C286 ◽

Cited By ~ 8

Author(s):

Guillermo J. Pérez ◽

Mayurika Desai ◽

Seth Anderson ◽

Fabiana S. Scornik

Keyword(s):

Membrane Potential ◽

Calcium Release ◽

Single Channel ◽

Calcium Influx ◽

Ryanodine Receptors ◽

Bk Channels ◽

Release Mechanism ◽

Dependent Manner ◽

Whole Cell ◽

Intracardiac Neurons

We studied principal neurons from canine intracardiac (IC) ganglia to determine whether large-conductance calcium-activated potassium (BK) channels play a role in their excitability. We performed whole cell recordings in voltage- and current-clamp modes to measure ion currents and changes in membrane potential from isolated canine IC neurons. Whole cell currents from these neurons showed fast- and slow-activated outward components. Both current components decreased in the absence of calcium and following 1–2 mM tetraethylammonium (TEA) or paxilline. These results suggest that BK channels underlie these current components. Single-channel analysis showed that BK channels from IC neurons do not inactivate in a time-dependent manner, suggesting that the dynamic of the decay of the fast current component is akin to that of intracellular calcium. Immunohistochemical studies showed that BK channels and type 2 ryanodine receptors are coexpressed in IC principal neurons. We tested whether BK current activation in these neurons occurred via a calcium-induced calcium release mechanism. We found that the outward currents of these neurons were not affected by the calcium depletion of intracellular stores with 10 mM caffeine and 10 μM cyclopiazonic acid. Thus, in canine intracardiac neurons, BK currents are directly activated by calcium influx. Membrane potential changes elicited by long (400 ms) current injections showed a tonic firing response that was decreased by TEA or paxilline. These data strongly suggest that the BK current present in canine intracardiac neurons regulates action potential activity and could increase these neurons excitability.

Download Full-text

Functional Triads Consisting of Ryanodine Receptors, Ca2+ Channels, and Ca2+-Activated K+ Channels in Bullfrog Sympathetic Neurons

The Journal of General Physiology ◽

10.1085/jgp.116.5.697 ◽

2000 ◽

Vol 116 (5) ◽

pp. 697-720 ◽

Cited By ~ 61

Author(s):

Tenpei Akita ◽

Kenji Kuba

Keyword(s):

High Speed ◽

Channel Blocker ◽

Ganglion Cells ◽

Sympathetic Neurons ◽

Ryanodine Receptors ◽

Bk Channels ◽

K Channel ◽

Sk Channels ◽

K Current ◽

Ca2 Channels

Fluorescent ryanodine revealed the distribution of ryanodine receptors in the submembrane cytoplasm (less than a few micrometers) of cultured bullfrog sympathetic ganglion cells. Rises in cytosolic Ca2+ ([Ca2+]i) elicited by single or repetitive action potentials (APs) propagated at a high speed (150 μm/s) in constant amplitude and rate of rise in the cytoplasm bearing ryanodine receptors, and then in the slower, waning manner in the deeper region. Ryanodine (10 μM), a ryanodine receptor blocker (and/or a half opener), or thapsigargin (1–2 μM), a Ca2+-pump blocker, or ω-conotoxin GVIA (ω-CgTx, 1 μM), a N-type Ca2+ channel blocker, blocked the fast propagation, but did not affect the slower spread. Ca2+ entry thus triggered the regenerative activation of Ca2+-induced Ca2+ release (CICR) in the submembrane region, followed by buffered Ca2+ diffusion in the deeper cytoplasm. Computer simulation assuming Ca2+ release in the submembrane region reproduced the Ca2+ dynamics. Ryanodine or thapsigargin decreased the rate of spike repolarization of an AP to 80%, but not in the presence of iberiotoxin (IbTx, 100 nM), a BK-type Ca2+-activated K+ channel blocker, or ω-CgTx, both of which decreased the rate to 50%. The spike repolarization rate and the amplitude of a single AP-induced rise in [Ca2+]i gradually decreased to a plateau during repetition of APs at 50 Hz, but reduced less in the presence of ryanodine or thapsigargin. The amplitude of each of the [Ca2+]i rise correlated well with the reduction in the IbTx-sensitive component of spike repolarization. The apamin-sensitive SK-type Ca2+-activated K+ current, underlying the afterhyperpolarization of APs, increased during repetitive APs, decayed faster than the accompanying rise in [Ca2+]i, and was suppressed by CICR blockers. Thus, ryanodine receptors form a functional triad with N-type Ca2+ channels and BK channels, and a loose coupling with SK channels in bullfrog sympathetic neurons, plastically modulating AP.

Download Full-text