Effects of Strontium on the Permeation and Gating Phenotype of Calcium Channels in Hair Cells

To minimize the effects of Ca2+ buffering and signaling, this study sought to examine single Ca2+ channel properties using Sr2+ ions, which substitute well for Ca2+ but bind weakly to intracellular Ca2+ buffers. Two single-channel fluctuations were distinguished by their sensitivity to dihydropyridine agonist (L-type) and insensitivity toward dihydropyridine antagonist (non-L-type). The L- and non-L-type single channels were observed with single-channel conductances of 16 and 19 pS at 70 mM Sr2+ and 11 and 13 pS at 5 mM Sr2+, respectively. We obtained KD estimates of 5.2 and 1.9 mM for Sr2+ for L- and non-L-type channels, respectively. At Ca2+ concentration of ∼2 mM, the single-channel conductances of Sr2+ for the L-type channel was ∼1.5 and 4.0 pS for the non-L-type channels. Thus the limits of single-channel microdomain at the membrane potential of a hair cell (e.g., −65 mV) for Sr2+ ranges from 800 to 2,000 ion/ms, assuming an ECa of 100 mV. The channels are ≥4-fold more sensitive at the physiological concentration ranges than at concentrations >10 mM. Additionally, the channels have the propensity to dwell in the closed state at high concentrations of Sr2+, which is reflected in the time constant of the first latency distributions. It is concluded that the concentration of the permeant ion modulates the gating of hair cell Ca2+ channels. Finally, the closed state/s that is/are altered by high concentrations of Sr2+ may represent divalent ion-dependent inactivation of the L-type channel.

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Caffeine-induced inhibition of inositol(1,4,5)-trisphosphate-gated calcium channels from cerebellum.

Molecular Biology of the Cell ◽

10.1091/mbc.5.1.97 ◽

1994 ◽

Vol 5 (1) ◽

pp. 97-103 ◽

Cited By ~ 57

Author(s):

I Bezprozvanny ◽

S Bezprozvannaya ◽

B E Ehrlich

Keyword(s):

Lipid Bilayers ◽

Inhibitory Action ◽

Single Channel ◽

Ryanodine Receptors ◽

Single Channels ◽

Ca Channels ◽

Open Time ◽

Inositol 1,4,5 Trisphosphate ◽

Insp3 Receptors ◽

Intracellular Ca

Effects of the xanthine drug caffeine on inositol (1,4,5)-trisphosphate (InsP3)-gated calcium (Ca) channels from canine cerebellum were studied using single channels incorporated into planar lipid bilayers. Caffeine, used widely as an agonist of ryanodine receptors, inhibited the activity of InsP3-gated Ca channels in a noncooperative fashion with half-inhibition at 1.64 mM caffeine. The frequency of channel openings was decreased more than threefold after addition of 5 mM caffeine; there was only a small effect on mean open time of the channels, and the single channel conductance was unchanged. Increased InsP3 concentration overcame the inhibitory action of caffeine, but caffeine did not reduce specific [3H]InsP3 binding to the receptor. The inhibitory action of caffeine on InsP3 receptors suggests that the action of caffeine on the intracellular Ca pool must be interpreted with caution when both ryanodine receptors and InsP3 receptors are present in the cell.

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Pathways of HERG inactivation

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1999.277.1.h199 ◽

1999 ◽

Vol 277 (1) ◽

pp. H199-H210 ◽

Cited By ~ 14

Author(s):

Johann Kiehn ◽

Antonio E. Lacerda ◽

Arthur M. Brown

Keyword(s):

Xenopus Oocytes ◽

Single Channel ◽

Inward Rectification ◽

Single Channels ◽

Channel Current ◽

Cardiac Action ◽

Current Voltage ◽

Inwardly Rectifying ◽

Channel Properties ◽

Current Voltage Relation

The rapid, repolarizing K+ current in cardiomyocytes ( I Kr) has unique inwardly rectifying properties that contribute importantly to the downstroke of the cardiac action potential. The human ether-à-go-go-related gene ( HERG) expresses a macroscopic current virtually identical to I Kr, but a description of the single-channel properties that cause rectification is lacking. For this reason we measured single-channel and macropatch currents heterologously expressed by HERG in Xenopus oocytes. Our experiments had two main findings. First, the single-channel current-voltage relation showed inward rectification, and conductance was 9.7 pS at −100 mV and 3.9 pS at 100 mV when measured in symmetrical 100 mM K+ solutions. Second, single channels frequently showed no openings during depolarization but nevertheless revealed bursts of openings during repolarization. This type of gating may explain the inward rectification of HERG currents. To test this hypothesis, we used a three-closed state kinetics model and obtained rate constants from fits to macropatch data. Results from the model are consistent with rapid inactivation from closed states as a significant source of HERG rectification.

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Voltage-dependent inactivation of T-tubular skeletal calcium channels in planar lipid bilayers.

The Journal of General Physiology ◽

10.1085/jgp.97.2.393 ◽

1991 ◽

Vol 97 (2) ◽

pp. 393-412 ◽

Cited By ~ 19

Author(s):

R Mejía-Alvarez ◽

M Fill ◽

E Stefani

Keyword(s):

Calcium Channels ◽

Lipid Bilayers ◽

Single Channel ◽

Channel Activity ◽

T Tube ◽

Dependent Inactivation ◽

Voltage Dependent ◽

Single Channel Activity ◽

Kinetics Of ◽

Channel Properties

Single-channel properties of dihydropyridine (DHP)-sensitive calcium channels isolated from transverse tubular (T-tube) membrane of skeletal muscle were explored. Single-channel activity was recorded in planar lipid bilayers after fusion of highly purified rabbit T-tube microsomes. Two populations of DHP-sensitive calcium channels were identified. One type of channel (noninactivating) was active (2 microM +/- Bay K 8644) at steady-state membrane potentials and has been studied in other laboratories. The second type of channel (inactivating) was transiently activated during voltage pulses and had a very low open probability (Po) at steady-state membrane potentials. Inactivating channel activity was observed in 47.3% of the experiments (n = 84 bilayers). The nonstationary kinetics of this channel was determined using a standard voltage pulse (HP = -50 mV, pulse to 0 mV). The time constant (tau) of channel activation was 23 ms. During the mV). The time constant (tau) of channel activation was 23 ms. During the pulse, channel activity decayed (inactivated) with a tau of 3.7 s. Noninactivating single-channel activity was well described by a model with two open and two closed states. Inactivating channel activity was described by the same model with the addition of an inactivated state as proposed for cardiac muscle. The single-channel properties were compared with the kinetics of DHP-sensitive inward calcium currents (ICa) measured at the cellular level. Our results support the hypothesis that voltage-dependent inactivation of single DHP-sensitive channels contributes to the decay of ICa.

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Etiology of the membrane potential of rat white fat adipocytes

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00446.2013 ◽

2014 ◽

Vol 307 (2) ◽

pp. E161-E175 ◽

Cited By ~ 8

Author(s):

Donna C. Bentley ◽

Pawitra Pulbutr ◽

Sue Chan ◽

Paul A. Smith

Keyword(s):

Membrane Potential ◽

Single Channel ◽

Reversal Potential ◽

Anion Channel ◽

Equilibrium Potential ◽

Physiological Processes ◽

Cation Permeability ◽

Intracellular Ca ◽

White Fat ◽

Channel Properties

The plasma membrane potential ( Vm) is key to many physiological processes; however, its ionic etiology in white fat adipocytes is poorly characterized. To address this question, we employed the perforated patch current clamp and cell-attached patch clamp methods in isolated primary white fat adipocytes and their cellular model 3T3-L1. The resting Vm of primary and 3T3-L1 adipocytes were −32.1 ± 1.2 mV ( n = 95) and −28.8 ± 1.2 mV ( n = 87), respectively. Vm was independent of cell size and fat content. Elevation of extracellular K+ to 50 mM by equimolar substitution of bath Na+ did not affect Vm, whereas substitution of bath Na+ with the membrane-impermeant cation N-methyl-d-glucamine+-hyperpolarized Vm by 16 mV, data indicative of a nonselective cation permeability. Substitution of 133 mM extracellular Cl− with gluconate-depolarized Vm by 25 mV, whereas Cl− substitution with I− caused a −9 mV hyperpolarization. Isoprenaline (10 μM), but not insulin (100 nM), significantly depolarized Vm. Single-channel ion activity was voltage independent; currents were indicative for Cl− with an inward slope conductance of 16 ± 1.3 pS ( n = 11) and a reversal potential close to the Cl− equilibrium potential, −29 ± 1.6 mV. Although the reduction of extracellular Cl− elevated the intracellular Ca2+ of adipocytes, this was not as large as that produced by elevation of extracellular K+. In conclusion, the Vm of white fat adipocytes is well described by the Goldman-Hodgkin-Katz equation with a predominant permeability to Cl−, where its biophysical and single-channel properties suggest a volume-sensitive anion channel identity. Consequently, changes in serum Cl− homeostasis or the adipocyte's permeability to this anion via drugs will affect its Vm, intracellular Ca2+, and ultimately its function and its role in metabolic control.

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An Investigation on the Performance of an Oxidation Catalyst Using Two-dimensional Simulation with Detailed Reaction Mechanism

Chemical Product and Process Modeling ◽

10.1515/cppm-2019-0115 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Sreeharsh Nair ◽

Mayank Mittal

Keyword(s):

Single Channel ◽

Catalytic Converter ◽

Oxidation Catalyst ◽

Low Temperature Combustion ◽

Wide Range ◽

Detailed Reaction Mechanism ◽

Temperature Combustion ◽

High Concentrations ◽

Dimensional Simulation ◽

Surface Reaction Kinetics

AbstractThe advent of stricter emission standards has increased the importance of aftertreatment devices and the role of numerical simulations in the evolution of better catalytic converters in order to satisfy these emission regulations. In this paper, a 2-D numerical simulation of a single channel of the monolith catalytic converter is presented by using detailed surface reaction kinetics aiming to investigate the chemical behaviour inside the converter. The model has been developed to study the conversion of carbon monoxide (CO) in the presence of propene (C3H6) for low-temperature combustion (LTC) engine application. The inhibition effect of C3H6 over a wide range of CO inlet concentrations is investigated. Considering both low and high levels of CO concentration at the inlet, the 2-D model predicted better results than their corresponding 1-D counterparts when compared with the experimental data from literature. It was also observed that C3H6 inhibition at high temperatures was significant, particularly for high concentrations of CO compared to low concentrations of CO at the inlet.

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cAMP-dependent regulation of IKs single-channel kinetics

The Journal of General Physiology ◽

10.1085/jgp.201611734 ◽

2017 ◽

Vol 149 (8) ◽

pp. 781-798 ◽

Cited By ~ 10

Author(s):

Emely Thompson ◽

Jodene Eldstrom ◽

Maartje Westhoff ◽

Donald McAfee ◽

Elise Balse ◽

...

Keyword(s):

Action Potential ◽

Single Channel ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Surface Expression ◽

Voltage Sensor ◽

Single Channels ◽

Adrenergic Stimulation ◽

Channel Opening ◽

Sensor Activation

The delayed potassium rectifier current, IKs, is composed of KCNQ1 and KCNE1 subunits and plays an important role in cardiac action potential repolarization. During β-adrenergic stimulation, 3′-5′-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) phosphorylates KCNQ1, producing an increase in IKs current and a shortening of the action potential. Here, using cell-attached macropatches and single-channel recordings, we investigate the microscopic mechanisms underlying the cAMP-dependent increase in IKs current. A membrane-permeable cAMP analog, 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP), causes a marked leftward shift of the conductance–voltage relation in macropatches, with or without an increase in current size. Single channels exhibit fewer silent sweeps, reduced first latency to opening (control, 1.61 ± 0.13 s; cAMP, 1.06 ± 0.11 s), and increased higher-subconductance-level occupancy in the presence of cAMP. The E160R/R237E and S209F KCNQ1 mutants, which show fixed and enhanced voltage sensor activation, respectively, largely abolish the effect of cAMP. The phosphomimetic KCNQ1 mutations, S27D and S27D/S92D, are much less and not at all responsive, respectively, to the effects of PKA phosphorylation (first latency of S27D + KCNE1 channels: control, 1.81 ± 0.1 s; 8-CPT-cAMP, 1.44 ± 0.1 s, P < 0.05; latency of S27D/S92D + KCNE1: control, 1.62 ± 0.1 s; cAMP, 1.43 ± 0.1 s, nonsignificant). Using total internal reflection fluorescence microscopy, we find no overall increase in surface expression of the channel during exposure to 8-CPT-cAMP. Our data suggest that the cAMP-dependent increase in IKs current is caused by an increase in the likelihood of channel opening, combined with faster openings and greater occupancy of higher subconductance levels, and is mediated by enhanced voltage sensor activation.

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Fluid pressure modulates L-type Ca2+ channel via enhancement of Ca2+-induced Ca2+ release in rat ventricular myocytes

AJP Cell Physiology ◽

10.1152/ajpcell.00381.2007 ◽

2008 ◽

Vol 294 (4) ◽

pp. C966-C976 ◽

Cited By ~ 22

Author(s):

Sunwoo Lee ◽

Joon-Chul Kim ◽

Yuhua Li ◽

Min-Jeong Son ◽

Sun-Hee Woo

Keyword(s):

Ventricular Myocytes ◽

Fluid Pressure ◽

Inhibitory Effect ◽

Cellular Mechanism ◽

Confocal Imaging ◽

Rat Ventricular Myocytes ◽

Whole Cell Patch Clamp ◽

Current Voltage ◽

Intracellular Ca ◽

High Concentrations

This study examines whether fluid pressure (FP) modulates the L-type Ca2+ channel in cardiomyocytes and investigates the underlying cellular mechanism(s) involved. A flow of pressurized (∼16 dyn/cm2) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes using a microperfusion method. The Ca2+ current ( ICa) and cytosolic Ca2+ signals were measured using a whole cell patch-clamp and confocal imaging, respectively. It was found that the FP reversibly suppressed ICa (by 25%) without altering the current-voltage relationships, and it accelerated the inactivation of ICa. The level of ICa suppression by FP depended on the level and duration of pressure. The Ba2+ current through the Ca2+ channel was only slightly decreased by the FP (5%), suggesting an indirect inhibition of the Ca2+ channel during FP stimulation. The cytosolic Ca2+ transients and the basal Ca2+ in field-stimulated ventricular myocytes were significantly increased by the FP. The effects of the FP on the ICa and on the Ca2+ transient were resistant to the stretch-activated channel inhibitors, GsMTx-4 and streptomycin. Dialysis of myocytes with high concentrations of BAPTA, the Ca2+ buffer, eliminated the FP-induced acceleration of ICa inactivation and reduced the inhibitory effect of the FP on ICa by ≈80%. Ryanodine and thapsigargin, abolishing sarcoplasmic reticulum Ca2+ release, eliminated the accelerating effect of FP on the ICa inactivation, and they reduced the inhibitory effect of FP on the ICa. These results suggest that the fluid pressure indirectly suppresses the Ca2+ channel by enhancing the Ca2+-induced intracellular Ca2+ release in rat ventricular myocytes.

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