Voltage dependence of the Ca2+-activated K+channel KCa3.1 in human erythroleukemia cells

2013 ◽  
Vol 304 (9) ◽  
pp. C858-C872 ◽  
Author(s):  
Colin J. Stoneking ◽  
Oshini Shivakumar ◽  
David Nicholson Thomas ◽  
William H. Colledge ◽  
Michael J. Mason
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
K Channel ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Erythroleukemia Cells ◽  
Voltage Dependent ◽  
Hel Cells ◽  
Cell Current ◽  
Whole Cell Current

We have isolated a K+-selective, Ca2+-dependent whole cell current and single-channel correlate in the human erythroleukemia (HEL) cell line. The whole cell current was inhibited by the intermediate-conductance KCa3.1 inhibitors clotrimazole, TRAM-34, and charybdotoxin, unaffected by the small-conductance KCa2 family inhibitor apamin and the large-conductance KCa1.1 inhibitors paxilline and iberiotoxin, and augmented by NS309. The single-channel correlate of the whole cell current was blocked by TRAM-34 and clotrimazole, insensitive to paxilline, and augmented by NS309 and had a single-channel conductance in physiological K+gradients of ∼9 pS. RT-PCR revealed that the KCa3.1 gene, but not the KCa1.1 gene, was expressed in HEL cells. The KCa3.1 current, isolated in HEL cells under whole cell patch-clamp conditions, displayed an activated current component during depolarizing voltage steps from hyperpolarized holding potentials and tail currents upon repolarization, consistent with voltage-dependent modulation. This activated current increased with increasing voltage steps above −40 mV and was sensitive to inhibition by clotrimazole, TRAM-34, and charybdotoxin and insensitive to apamin, paxilline, and iberiotoxin. In single-channel experiments, depolarization resulted in an increase in open channel probability ( Po) of KCa3.1, with no increase in channel number. The voltage modulation of Powas an increasing monotonic function of voltage. In the absence of elevated Ca2+, voltage was ineffective at inducing channel activity in whole cell and single-channel experiments. These data indicate that KCa3.1 in HEL cells displays a unique form of voltage dependence modulating Po.

scholarly journals Volume-Dependent Atp-Conductive Large-Conductance Anion Channel as a Pathway for Swelling-Induced Atp Release

2001 ◽  
Vol 118 (3) ◽  
pp. 251-266 ◽  
Author(s):  
Ravshan Z. Sabirov ◽  
Amal K. Dutta ◽  
Yasunobu Okada
Keyword(s):  
Single Channel ◽  
Atp Release ◽  
Anion Channel ◽  
Time Dependent ◽  
Whole Cell ◽  
Dependent Inactivation ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Cell Current ◽  
Whole Cell Current

In mouse mammary C127i cells, during whole-cell clamp, osmotic cell swelling activated an anion channel current, when the phloretin-sensitive, volume-activated outwardly rectifying Cl− channel was eliminated. This current exhibited time-dependent inactivation at positive and negative voltages greater than around ±25 mV. The whole-cell current was selective for anions and sensitive to Gd3+. In on-cell patches, single-channel events appeared with a lag period of ∼15 min after a hypotonic challenge. Under isotonic conditions, cell-attached patches were silent, but patch excision led to activation of currents that consisted of multiple large-conductance unitary steps. The current displayed voltage- and time-dependent inactivation similar to that of whole-cell current. Voltage-dependent activation profile was bell-shaped with the maximum open probability at −20 to 0 mV. The channel in inside-out patches had the unitary conductance of ∼400 pS, a linear current-voltage relationship, and anion selectivity. The outward (but not inward) single-channel conductance was suppressed by extracellular ATP with an IC50 of 12.3 mM and an electric distance (δ) of 0.47, whereas the inward (but not outward) conductance was inhibited by intracellular ATP with an IC50 of 12.9 mM and δ of 0.40. Despite the open channel block by ATP, the channel was ATP-conductive with PATP/PCl of 0.09. The single-channel activity was sensitive to Gd3+, SITS, and NPPB, but insensitive to phloretin, niflumic acid, and glibenclamide. The same pharmacological pattern was found in swelling-induced ATP release. Thus, it is concluded that the volume- and voltage-dependent ATP-conductive large-conductance anion channel serves as a conductive pathway for the swelling-induced ATP release in C127i cells.

scholarly journals Cell swelling activates ATP-dependent voltage-gated chloride channels in M-1 mouse cortical collecting duct cells.

1996 ◽  
Vol 108 (3) ◽  
pp. 177-193 ◽  
Author(s):  
K Meyer ◽  
C Korbmacher
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Collecting Duct ◽  
High Energy ◽  
Cell Swelling ◽  
Single Channels ◽  
Cortical Collecting Duct ◽  
Whole Cell ◽  
Cell Current ◽  
Whole Cell Current

In the present study we used whole-cell patch clamp recordings to investigate swelling-activated Cl-currents (ICl-swell) in M-1 mouse cortical collecting duct (CCD) cells. Hypotonic cell swelling reversibly increased the whole-cell Cl- conductance by about 30-fold. The I-V relationship was outwardly-rectifying and ICl-swell displayed a characteristic voltage-dependence with relatively fast inactivation upon large depolarizing and slow activation upon hyperpolarizing voltage steps. Reversal potential measurements revealed a selectivity sequence SCN- > I- > Br- > Cl- > > gluconate. ICl-swell was inhibited by tamoxifen, NPPB (5-nitro-2(3-phenylpropylamino)-benzoate), DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid), flufenamic acid, niflumic acid, and glibenclamide, in descending order of potency. Extracellular cAMP had no significant effect. ICl-swell was Ca2+ independent, but current activation depended on the presence of a high-energy gamma-phosphate group from intracellular ATP or ATP gamma S. Moreover, it depended on the presence of intracellular Mg2+ and was inhibited by staurosporine, which indicates that a phosphorylation step is involved in channel activation. Increasing the cytosolic Ca2+ concentration by using ionomycin stimulated Cl- currents with a voltage dependence different from that of ICl-swell. Analysis of whole-cell current records during early onset of ICl-swell and during final recovery revealed discontinuous step-like changes of the whole-cell current level which were not observed under nonswelling conditions. A single-channel I-V curve constructed using the smallest resolvable current transitions detected at various holding potentials and revealed a slope conductance of 55, 15, and 8 pS at +120, 0, and -120 mV, respectively. The larger current steps observed in these recordings had about 2, 3, or 4 times the size of the putative single-channel current amplitude, suggesting a coordinated gating of several individual channels or channel subunits. In conclusion we have functionally characterized ICl-swell in M-1 CCD cells and have identified the underlying single channels in whole-cell current recordings.

scholarly journals The Effectiveness in Activating M-Type K+ Current Produced by Solifenacin ([(3R)-1-azabicyclo[2.2.2]octan-3-yl] (1S)-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carboxylate): Independent of Its Antimuscarinic Action

2021 ◽  
Vol 22 (22) ◽  
pp. 12399
Author(s):  
Hsin-Yen Cho ◽  
Tzu-Hsien Chuang ◽  
Sheng-Nan Wu
Keyword(s):  
Single Channel ◽  
Ionic Currents ◽  
Reaction Scheme ◽  
Gh3 Cells ◽  
Whole Cell ◽  
Type K ◽  
Cell Current ◽  
K Current ◽  
Subsequent Addition ◽  
Whole Cell Current

Solifenacin (Vesicare®, SOL), known to be a member of isoquinolines, is a muscarinic antagonist that has anticholinergic effect, and it has been beneficial in treating urinary incontinence and neurogenic detrusor overactivity. However, the information regarding the effects of SOL on membrane ionic currents is largely uncertain, despite its clinically wide use in patients with those disorders. In this study, the whole-cell current recordings revealed that upon membrane depolarization in pituitary GH3 cells, the exposure to SOL concentration-dependently increased the amplitude of M-type K+ current (IK(M)) with effective EC50 value of 0.34 μM. The activation time constant of IK(M) was concurrently shortened in the SOL presence, hence yielding the KD value of 0.55 μM based on minimal reaction scheme. As cells were exposed to SOL, the steady-state activation curve of IK(M) was shifted along the voltage axis to the left with no change in the gating charge of the current. Upon an isosceles-triangular ramp pulse, the hysteretic area of IK(M) was increased by adding SOL. As cells were continually exposed to SOL, further application of acetylcholine (1 μM) failed to modify SOL-stimulated IK(M); however, subsequent addition of thyrotropin releasing hormone (TRH, 1 μM) was able to counteract SOL-induced increase in IK(M) amplitude. In cell-attached single-channel current recordings, bath addition of SOL led to an increase in the activity of M-type K+ (KM) channels with no change in the single channel conductance; the mean open time of the channel became lengthened. In whole-cell current-clamp recordings, the SOL application reduced the firing of action potentials (APs) in GH3 cells; however, either subsequent addition of TRH or linopirdine was able to reverse SOL-mediated decrease in AP firing. In hippocampal mHippoE-14 neurons, the IK(M) was also stimulated by adding SOL. Altogether, findings from this study disclosed for the first time the effectiveness of SOL in interacting with KM channels and hence in stimulating IK(M) in electrically excitable cells, and this noticeable action appears to be independent of its antagonistic activity on the canonical binding to muscarinic receptors expressed in GH3 or mHippoE-14 cells.

Basolateral K+ conductance in principal cells of rat CCD

2005 ◽  
Vol 288 (3) ◽  
pp. F493-F504 ◽  
Author(s):  
Daniel A. Gray ◽  
Gustavo Frindt ◽  
Yu-Yang Zhang ◽  
Lawrence G. Palmer
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
Whole Cell ◽  
Principal Cells ◽  
Luminal Membrane ◽  
High K ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
Cell Current ◽  
Single Channel Currents

Whole cell K+ current was measured by forming seals on the luminal membrane of principal cells in split-open rat cortical collecting ducts. The mean inward, Ba2+-sensitive conductance, with 40 mM extracellular K+, was 76 ± 12 and 141 ± 22 nS/cell for animals on control and high-K+ diets, respectively. The apical contribution to this was estimated to be 3 and 16 nS/cell on control and high-K+ diets, respectively. To isolate the basolateral component of whole cell current, we blocked ROMK channels with either tertiapin-Q or intracellular acidification to pH 6.6. The current was weakly inward rectifying when bath K+ was ≥40 mM but became more strongly rectified when bath K+ was lowered into the physiological range. Including 1 mM spermine in the pipette moderately increased rectification, but most of the outward current remained. The K+ current did not require intracellular Ca2+ and was not inhibited by 3 mM ATP in the pipette. The negative log of the acidic dissociation constant (p Ka) was ∼6.5. Block by extracellular Ba2+ was voltage dependent with apparent Ki at −40 and −80 mV of ∼160 and ∼80 μM, respectively. The conductance was TEA insensitive. Substitution of Rb+ or NH4+ for K+ led to permeability ratios of 0.65 ± 0.07 and 0.15 ± 0.02 and inward conductance ratios of 0.17 ± 0.03 and 0.57 ± 0.09, respectively. Analysis of Ba2+-induced noise, with 40 mM extracellular K+, yielded single-channel currents of 0.39 ± 0.04 and −0.28 ± 0.04 pA at voltages of 0 and −40 mV, respectively, and a single-channel conductance of 17 ± 1 pS.

scholarly journals Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units.

1996 ◽  
Vol 107 (6) ◽  
pp. 695-714 ◽  
Author(s):  
E H Larsen ◽  
S E Gabriei ◽  
M J Stutts ◽  
J Fullton ◽  
E M Price ◽  
...  
Keyword(s):  
Chloride Channels ◽  
Single Channel ◽  
Low Frequency ◽  
Power Density Spectrum ◽  
Current Fluctuations ◽  
Sf9 Cells ◽  
Density Spectrum ◽  
Whole Cell ◽  
Cell Current ◽  
Whole Cell Current

The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result from simultaneous open/shut events of two or more channel units.

Effects of Spike Parameters and Neuromodulators on Action Potential Waveform-Induced Calcium Entry Into Pyramidal Neurons

2001 ◽  
Vol 85 (4) ◽  
pp. 1412-1423 ◽  
Author(s):  
Ansalan E. Stewart ◽  
Robert C. Foehring
Keyword(s):  
Charge Carrier ◽  
Action Potential ◽  
Calcium Channel ◽  
Pyramidal Neurons ◽  
Total Charge ◽  
Whole Cell ◽  
Voltage Dependent ◽  
Cell Current ◽  
Whole Cell Current ◽  
Neocortical Pyramidal Neurons

Neocortical pyramidal neurons express several different calcium channel types. Previous studies with square voltage steps have found modest biophysical differences between these calcium channel types as well as differences in their modulation by transmitters. We used acutely dissociated neocortical pyramidal neurons to test whether this diversity extends to different activation by physiological stimuli. We conclude that 1) peak amplitude, latency to peak, and the total charge entry for the Ca2+ channel current is dependent on the shape of the mock action potential waveforms (APWs). 2) The percent contribution of the five high-voltage-activated currents to the whole cell current was not altered by using an APW as opposed to a voltage step to elicit the current. 3) The identity of the charge carrier affects the amplitude and decay of the whole cell current. With Ca2+, there was a greater contribution of T-type current to the whole cell current. 4) Total Ba2+ charge entry is linearly dependent on the number of spikes in the stimulating waveform and relatively insensitive to spike frequency. 5) Current decay was greatest with Ca2+ as the charge carrier and with minimal internal chelation. 6) Voltage-dependent neurotransmitter-mediated modulations can be reversed by multiple spikes. The extent of the reversal is dependent on the number of spikes in the stimulating waveform. Thus the neuronal activity pattern can determine the effectiveness of voltage-dependent and -independent modulatory pathways in neocortical pyramidal neurons.

Voltage-dependent block by neomycin of the ATP-induced whole cell current of guinea-pig outer hair cells

1993 ◽  
Vol 70 (4) ◽  
pp. 1593-1605 ◽  
Author(s):  
X. Lin ◽  
R. I. Hume ◽  
A. L. Nuttall
Keyword(s):  
Steady State ◽  
Guinea Pig ◽  
Hair Cells ◽  
Time Course ◽  
Outer Hair Cells ◽  
Current Noise ◽  
Whole Cell ◽  
Voltage Dependent ◽  
Cell Current ◽  
Whole Cell Current

1. The effects of externally applied ATP and neomycin on whole cell currents of isolated guinea pig cochlear outer hair cells (OHCs) were studied using the whole cell voltage-clamp technique. In OCHs held at -70 mV, ATP activated a large inward current. In the presence of neomycin, the ATP-induced whole cell current activated along a relatively unaltered time course, but the current then decreased to a reduced steady level. The neomycin inhibition of the ATP-induced current was dose dependent. The half-inhibitory concentration (IC50) of neomycin measured at steady state was estimated to be 90 microM. 2. Neomycin inhibition of the ATP response could not be reversed by increasing the concentration of ATP, indicating that the effect was noncompetitive. The inhibition was voltage dependent and was greatly reduced when OHCs were held at positive potentials. 3. Cells treated with 100 microM ATP gave maximal current responses. Addition of neomycin substantially increased membrane current noise of the 100 microM ATP responses. When neomycin concentration was varied from 10 to 500 microM, the current noise level peaked between 50 and 100 microM. The noise increase was observed at negative holding potentials but not at positive potentials. 4. The neomycin-induced whole cell current noise was used to estimate the size of the underlying elementary current. The ATP-induced single channel current of OHCs at -70 mV was estimated to be approximately 0.3 pA. The number of ATP-activated channels in a single OHC was estimated to be in the range of a few thousand. 5. The characteristics of the neomycin inhibition of ATP-induced currents were consistent with an open channel blocking mechanism. Analysis of the voltage dependence of the steady state neomycin inhibition suggested a neomycin binding site at an electrical distance of 0.3 from the extracellular side.

Voltage-dependent aminoglycoside blockade of the sarcoplasmic reticulum K+ channel

1986 ◽  
Vol 250 (3) ◽  
pp. C361-C364 ◽  
Author(s):  
Y. Oosawa ◽  
M. Sokabe
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Voltage Dependence ◽  
Single Channel ◽  
Phospholipid Bilayer ◽  
K Channel ◽  
Dependent Manner ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Voltage Dependent ◽  
The Way

Single-channel conductance of the K+ channel from sarcoplasmic reticulum (SR) was reduced by aminoglycoside antibiotics such as neomycin and ribostamycin and also by n-hexylamine from either side of the membrane in a dose- and voltage-dependent manner. K+ channels were incorporated into an artificial phospholipid bilayer. This inhibition follows a single-site titration curve. The voltage dependence of the inhibition is explained by assuming that these drugs bind to the open state of a single channel on one site located approximately 40% of the way through the membrane from the cis side (the side to which SR vesicles are added) when drugs are added to the cis side and bind on another site located approximately 40% of the way through the membrane from the trans side (the opposite side to the cis side) when drugs are added to the trans side.

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