scholarly journals L-type Ca2+ channels access multiple open states to produce two components of Bay K 8644-dependent current in GH3 cells.

1996 ◽  
Vol 108 (1) ◽  
pp. 13-26 ◽  
Author(s):  
D M Fass ◽  
E S Levitan
Keyword(s):  
Single Channel ◽  
Slow Component ◽  
Bay K 8644 ◽  
Gh3 Cells ◽  
Whole Cell ◽  
Tail Current ◽  
Current Decay ◽  
Cell Functions ◽  
Time Courses ◽  
Open States

To determine the number of L-channel populations responsible for producing the two components of whole-cell L-type Ca2+ channel current revealed by Bay K 8644 (Fass, D.M., and E.S. Levitan. 1996. J. Gen. Physiol. 108:1-11), L-type Ca2+ channel activity was recorded in cell-attached patches. Ensemble tail currents from most (six out of nine) single-channel patches had double-exponential time courses, with time constants that were similar to whole-cell tail current decay values. Also, in single-channel patches subjected to two different levels of depolarization, ensemble tail currents exactly reproduced the voltage dependence of activation of the two whole-cell components: The slow component is activated at more negative potentials than the fast component. In addition, deactivation of Bay K 8644-modified whole-cell L-current was slower after long (100-ms) depolarizations than after short (20-ms) depolarizations, and this phenomenon was also evident in ensemble tail currents from single L-channels. Thus, a single population of L-channels can produce the two components of macroscopic L-current deactivation. To determine how individual L-channels produce multiple macroscopic tail current components, we constructed ensemble tail currents from traces that contained a single opening upon repolarization and no reopenings. These ensemble tails were biexponential. This type of analysis also revealed that reopenings do not contribute to the slowing of tail current deactivation after long depolarizations. Thus, individual L-channels must have access to several open states to produce multiple macroscopic current components. We also obtained evidence that access to these open states can vary over time. Use of several open states may give L-channels the flexibility to participate in many cell functions.

scholarly journals Effective Activation of BKCa Channels by QO-40 (5-(Chloromethyl)-3-(Naphthalen-1-yl)-2-(Trifluoromethyl)Pyrazolo [1,5-a]pyrimidin-7(4H)-one), Known to Be an Opener of KCNQ2/Q3 Channels

2021 ◽  
Vol 14 (5) ◽  
pp. 388
Author(s):  
Wei-Ting Chang ◽  
Sheng-Nan Wu
Keyword(s):  
Single Channel ◽  
Slow Component ◽  
Ionic Channel ◽  
Gh3 Cells ◽  
Bkca Channel ◽  
Bkca Channels ◽  
Gating Charge ◽  
Ramp Voltage ◽  
K Current ◽  
The Mean

QO-40 (5-(chloromethyl)-3-(naphthalene-1-yl)-2-(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-7(4H)-one) is a novel and selective activator of KCNQ2/KCNQ3 K+ channels. However, it remains largely unknown whether this compound can modify any other type of plasmalemmal ionic channel. The effects of QO-40 on ion channels in pituitary GH3 lactotrophs were investigated in this study. QO-40 stimulated Ca2+-activated K+ current (IK(Ca)) with an EC50 value of 2.3 μM in these cells. QO-40-stimulated IK(Ca) was attenuated by the further addition of GAL-021 or paxilline but not by linopirdine or TRAM-34. In inside-out mode, this compound added to the intracellular leaflet of the detached patches stimulated large-conductance Ca2+-activated K+ (BKCa) channels with no change in single-channel conductance; however, there was a decrease in the slow component of the mean closed time of BKCa channels. The KD value required for the QO-40-mediated decrease in the slow component at the mean closure time was 1.96 μM. This compound shifted the steady-state activation curve of BKCa channels to a less positive voltage and decreased the gating charge of the channel. The application of QO-40 also increased the hysteretic strength of BKCa channels elicited by a long-lasting isosceles-triangular ramp voltage. In HEK293T cells expressing α-hSlo, QO-40 stimulated BKCa channel activity. Overall, these findings demonstrate that QO-40 can interact directly with the BKCa channel to increase the amplitude of IK(Ca) in GH3 cells.

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

1994 ◽  
Vol 71 (5) ◽  
pp. 1873-1882 ◽  
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.

scholarly journals Single calcium channel behavior in native skeletal muscle.

1995 ◽  
Vol 105 (2) ◽  
pp. 227-247 ◽  
Author(s):  
R T Dirksen ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Calcium Channels ◽  
Single Channel ◽  
Bay K 8644 ◽  
Dependent Manner ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Whole Cell ◽  
Macroscopic Properties ◽  
Voltage Dependent

The purpose of this study was to use whole-cell and cell-attached patches of cultured skeletal muscle myotubes to study the macroscopic and unitary behavior of voltage-dependent calcium channels under similar conditions. With 110 mM BaCl2 as the charge carrier, two types of calcium channels with markedly different single-channel and macroscopic properties were found. One class was DHP-insensitive, had a single-channel conductance of approximately 9 pS, yielded ensembles that displayed an activation threshold near -40 mV, and activated and inactivated rapidly in a voltage-dependent manner (T current). The second class could only be well resolved in the presence of the DHP agonist Bay K 8644 (5 microM) and had a single-channel conductance of approximately 14 pS (L current). The 14-pS channel produced ensembles exhibiting a threshold of approximately -10 mV that activated slowly (tau act approximately 20 ms) and displayed little inactivation. Moreover, the DHP antagonist, (+)-PN 200-110 (10 microM), greatly increased the percentage of null sweeps seen with the 14-pS channel. The open probability versus voltage relationship of the 14-pS channel was fitted by a Boltzmann distribution with a VP0.5 = 6.2 mV and kp = 5.3 mV. L current recorded from whole-cell experiments in the presence of 110 mM BaCl2 + 5 microM Bay K 8644 displayed similar time- and voltage-dependent properties as ensembles of the 14-pS channel. Thus, these data are the first comparison under similar conditions of the single-channel and macroscopic properties of T current and L current in native skeletal muscle, and identify the 9- and 14-pS channels as the single-channel correlates of T current and L current, respectively.

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.

Effects of dihydropyridine calcium channel modulators on cardiac sodium channels

1988 ◽  
Vol 254 (1) ◽  
pp. H140-H147 ◽  
Author(s):  
A. Yatani ◽  
D. L. Kunze ◽  
A. M. Brown
Keyword(s):  
Calcium Channels ◽  
Sodium Channels ◽  
Single Channel ◽  
Bay K 8644 ◽  
Sodium Currents ◽  
Whole Cell ◽  
Blocking Effects ◽  
Voltage Dependent ◽  
Cardiac Sodium Channels ◽  
Single Channel Currents

To investigate whether cardiac sodium channels have dihydropyridine (DHP) receptors we studied the effects of the optically pure (greater than 95%) enantiomers of the DHPs PN200–110 and BAY-K 8644 and the racemic DHP nitrendipine (NTD). Whole cell and single-channel sodium currents were recorded from cultured ventricular cells of neonatal rats using the patch-clamp method. NTD reduced cardiac sodium currents in a voltage-dependent manner. Inhibitory effects were due to an increase in traces without activity. The unit conductance remained unchanged. At negative holding potentials, NTD transiently increased the probability of channel opening. Both (+) and (-) PN 200–110 blocked sodium channels, although the (-) isomer was about one order of magnitude less effective. The blocking effects were voltage dependent. (+) BAY-K 8644 had similar blocking effects. (-) BAY-K 8644 produced an increase in sodium currents due to an increased frequency of channel openings and a marked prolongation of open time without any significant change in unit conductance. The DHPs have effects on cardiac sodium whole cell and single-channel currents that appear identical to and are as stereospecific as their effects on cardiac calcium currents, although the concentrations required are larger. In contrast the inwardly rectifying potassium channel (IK1) is unaffected by these DHPs. We conclude that functionally equivalent DHP receptors are present in cardiac sodium and calcium channels but not potassium channels and take this as evidence of the homology between sodium and calcium channels.

Effect of compartmentalized Ca2+ ions on electrical bursting activity of pancreatic beta-cells

1990 ◽  
Vol 258 (5) ◽  
pp. C955-C965 ◽  
Author(s):  
T. R. Chay
Keyword(s):  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Single Channel ◽  
Slow Component ◽  
Rapid Change ◽  
Outward Current ◽  
Plateau Phase ◽  
Whole Cell ◽  
Dependent Inactivation ◽  
Ca2 Channels

Patch-clamp single-channel and whole cell recordings have revealed new insights into the ionic channel properties in the pancreatic beta-cells. I have modeled the electrical events during the burst activity based on the observations that 1) the whole cell Ca2+ current has two functionally distinct components (fast and slow), 2) a fast component is inhibited by intracellular Ca2+, 3) a slow component is inactivated by depolarization, and 4) a significant fraction of the outward current is carried by the Ca2(+)-sensitive, voltage-gated K+ channels [K(Ca, V) channels]. The model contains a feature that the Ca2+ concentration in the submembrane compartment ([Ca2+]s) is higher than that in the cellular phase. At the plateau phase, [Ca2+]s is high enough to activate the K(Ca, V) channels. In addition to the K(Ca, V) channels, the model contains a voltage-activated Ca2+ channel that is quickly blocked by Ca2+ and slowly inhibited by voltage. Because the Ca2+ channel has an intracellular Ca2(+)-dependent inactivation gate, the increase in [Ca2+]s can inactivate the Ca2+ channels. According to this model, the spikes during the plateau phase are caused by a rapid movement of Ca2+ into and out of the compartment. Because of a rapid change in [Ca2+]s, the two competing currents, ICa and IK(Ca, V), fluctuate rapidly; the fluctuation leads to an emergence of spikes. The slow underlying wave is due to a voltage-dependent inactivation gate of the Ca2+ channels, which slowly closes as a result of depolarization. This model differs radically from my previous models, which featured a slowly varying intracellular Ca2+ concentration that was responsible for the underlying slow wave. Although the previous models give plateau fractions (the ratio between the plateau duration and cyclic time) to be far less than unity, the present model is the first of its kind that allows plateau fractions to be in the near-unity range.

scholarly journals Inactivation viewed through single sodium channels.

1984 ◽  
Vol 84 (4) ◽  
pp. 535-564 ◽  
Author(s):  
C A Vandenberg ◽  
R Horn
Keyword(s):  
Rate Constant ◽  
Rate Constants ◽  
Time Course ◽  
Voltage Dependence ◽  
Single Channel ◽  
Gh3 Cells ◽  
Activation Process ◽  
Whole Cell ◽  
Open Time ◽  
Voltage Dependent

Recordings of the sodium current in tissue-cultured GH3 cells show that the rate of inactivation in whole cell and averaged single channel records is voltage dependent: tau h varied e-fold/approximately 26 mV. The source of this voltage dependence was investigated by examining the voltage dependence of individual rate constants, estimated by maximum likelihood analysis of single channel records, in a five-state kinetic model. The rate constant for inactivating from the open state, rather than closing, increased with depolarization, as did the probability that an open channel inactivates. The rate constant for closing from the open state had the opposite voltage dependence. Both rate constants contributed to the mean open time, which was not very voltage dependent. Both open time and burst duration were less than tau h for voltages up to -20 mV. The slowest time constant of activation, tau m, was measured from whole cell records, by fitting a single exponential either to tail currents or to activating currents in trypsin-treated cells, in which the inactivation was abolished. tau m was a bell-shaped function of voltage and had a voltage dependence similar to tau h at voltages more positive than -35 mV, but was smaller than tau h. At potentials more negative than about -10 mV, individual channels may open and close several times before inactivating. Therefore, averaged single channel records, which correspond with macroscopic current elicited by a depolarization, are best described by a convolution of the first latency density with the autocorrelation function rather than with 1 - (channel open time distribution). The voltage dependence of inactivation from the open state, in addition to that of the activation process, is a significant factor in determining the voltage dependence of macroscopic inactivation. Although the rates of activation and inactivation overlapped greatly, independent and coupled inactivation could not be statistically distinguished for two models examined. Although rates of activation affect the observed rate of inactivation at intermediate voltages, extrapolation of our estimates of rate constants suggests that at very depolarized voltages the activation process is so fast that it is an insignificant factor in the time course of inactivation. Prediction of gating currents shows that an inherently voltage-dependent inactivation process need not produce a conspicuous component in the gating current.

scholarly journals The Function of Mitochondrial Calcium Uniporter at the Whole-Cell and Single Mitochondrion Levels in WT, MICU1 KO, and MICU2 KO Cells

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1520 ◽  
Author(s):  
Syed Islamuddin Shah ◽  
Ghanim Ullah
Keyword(s):  
Mitochondrial Function ◽  
Single Channel ◽  
Intact Cells ◽  
Whole Cell ◽  
Cell Functions ◽  
Calcium Uniporter ◽  
Function Change ◽  
Wide Range ◽  
Single Mitochondrion

Mitochondrial Ca2+ ([Ca2+]M) uptake through its Ca2+ uniporter (MCU) is central to many cell functions such as bioenergetics, spatiotemporal organization of Ca2+ signals, and apoptosis. MCU activity is regulated by several intrinsic proteins including MICU1, MICU2, and EMRE. While significant details about the role of MICU1, MICU2, and EMRE in MCU function have emerged recently, a key challenge for the future experiments is to investigate how these regulatory proteins modulate mitochondrial Ca2+ influx through MCU in intact cells under pathophysiological conditions. This is further complicated by the fact that several variables affecting MCU function change dynamically as cell functions. To overcome this void, we develop a data-driven model that closely replicates the behavior of MCU under a wide range of cytosolic Ca2+ ([Ca2+]C), [Ca2+]M, and mitochondrial membrane potential values in WT, MICU1 knockout (KO), and MICU2 KO cells at the single mitochondrion and whole-cell levels. The model is extended to investigate how MICU1 or MICU2 KO affect mitochondrial function. Moreover, we show how Ca2+ buffering proteins, the separation between mitochondrion and Ca2+-releasing stores, and the duration of opening of Ca2+-releasing channels affect mitochondrial function under different conditions. Finally, we demonstrate an easy extension of the model to single channel function of MCU.

scholarly journals Regulation of K+ Flow by a Ring of Negative Charges in the Outer Pore of BKCa Channels. Part II

2004 ◽  
Vol 124 (2) ◽  
pp. 185-197 ◽  
Author(s):  
Trude Haug ◽  
Riccardo Olcese ◽  
Ligia Toro ◽  
Enrico Stefani
Keyword(s):  
Single Channel ◽  
Slow Component ◽  
Ionic Current ◽  
K Channel ◽  
Gating Current ◽  
Wild Type ◽  
Open Probability ◽  
Activation Curve ◽  
Bkca Channels ◽  
Open States

Neutralization of the aspartate near the selectivity filter in the GYGD pore sequence (D292N) of the voltage- and Ca2+-activated K+ channel (MaxiK, BKCa) does not prevent conduction like the corresponding mutation in Shaker channel, but profoundly affects major biophysical properties of the channel (Haug, T., D. Sigg, S. Ciani, L. Toro, E. Stefani, and R. Olcese. 2004. J. Gen. Physiol. 124:173–184). Upon depolarizations, the D292N mutant elicited mostly gating current, followed by small or no ionic current, at voltages where the wild-type hSlo channel displayed robust ionic current. In fact, while the voltage dependence of the gating current was not significantly affected by the mutation, the overall activation curve was shifted by ∼20 mV toward more depolarized potentials. Several lines of evidence suggest that the mutation prevents population of certain open states that in the wild type lead to high open probability. The activation curves of WT and D292N can both be fitted to the sum of two Boltzmann distributions with identical slope factors and half activation potentials, just by changing their relative amplitudes. The steeper and more negative component of the activation curve was drastically reduced by the D292N mutation (from 0.65 to 0.30), suggesting that the population of open states that occurs early in the activation pathway is reduced. Furthermore, the slow component of the gating current, which has been suggested to reflect transitions from closed to open states, was greatly reduced in D292N channels. The D292N mutation also affected the limiting open probability: at 0 mV, the limiting open probability dropped from ∼0.5 for the wild-type channel to 0.06 in D292N (in 1 mM [Ca2+]i). In addition to these effects on gating charge and open probability, as already described in Part I, the D292N mutation introduces a ∼40% reduction of outward single channel conductance, as well as a strong outward rectification.

scholarly journals Nonmodal gating of cardiac calcium channels as revealed by dihydropyridines.

1989 ◽  
Vol 93 (6) ◽  
pp. 1243-1273 ◽  
Author(s):  
A E Lacerda ◽  
A M Brown
Keyword(s):  
Calcium Channels ◽  
Single Channel ◽  
Calcium Currents ◽  
Neonatal Rat ◽  
Whole Cell ◽  
Frequency Distributions ◽  
Time Frequency ◽  
Channel Current ◽  
Open Time ◽  
Open States

The hypothesis that dihydropyridine (DHP)-sensitive calcium channels have three distinct modes of gating has been examined. The major prediction is that the relative frequencies among modes depend on DHP concentration while the kinetics within a mode do not. We tested this by studying whole-cell and single-channel calcium currents in neonatal rat and adult guinea pig cardiac myocytes in different concentrations of several DHPs. In the absence of DHPs calcium currents declined with time but the kinetics, which are the focus of this study, were unchanged. Open-time frequency distributions had insignificant numbers of prolonged openings and were well fit by single tau's. Agonist DHP stereoisomers produced concentration-dependent changes in whole-cell tail current tau's. The frequency distribution of single calcium channel current open times became biexponential and the tau's were concentration dependent. The average number of openings per trace of channels with customary open times increased with increases in DHP concentration. Latencies to first opening for the customary openings and for prolonged openings were shorter in the presence of DHPs. A second larger conductance is another important feature of DHP-bound single calcium channels. Thus DHPs not only caused prolonged openings; they produced numerous changes in the kinetics of customary openings and increased channel conductance. It follows that these effects of DHPs do not support the hypothesis of modal gating of calcium channels. The mode model is not the only model excluded by the results; models in which DHPs are allowed to act only or mainly on open states are excluded, as are models in which the effects are restricted to inactivated states. We suggest a different type of model in which cooperative binding of DHPs at two sites produces the essential changes in kinetics and conductance.

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