scholarly journals Intrinsic gating of inward rectifier in bovine pulmonary artery endothelial cells in the presence or absence of internal Mg2+.

1990 ◽  
Vol 96 (1) ◽  
pp. 109-133 ◽  
Author(s):  
M R Silver ◽  
T E DeCoursey
Keyword(s):  
Endothelial Cells ◽  
Steady State ◽  
Pulmonary Artery ◽  
Voltage Dependence ◽  
Reversal Potential ◽  
Inward Rectifier ◽  
Patch Clamp Technique ◽  
Gating Mechanism ◽  
Boltzmann Function ◽  
Gating Process

Inward rectifier (IR) currents were studied in bovine pulmonary artery endothelial cells in the whole-cell configuration of the patch-clamp technique with extracellular K+ concentrations, [K+]o, ranging from 4.5 to 160 mM. Whether the concentration of free Mg2+ in the intracellular solution, [Mg2+]i, was 1.9 mM or nominally 0, the IR exhibited voltage- and time-dependent gating. The IR conductance was activated by hyperpolarization and deactivated by depolarization. Small steady-state outward IR currents were present up to approximately 40 mV more positive than the K+ reversal potential, EK, regardless of [Mg2+]i. Modeled as a first-order C in equilibrium O gating process, both the opening rate, alpha, and the closing rate, beta, were exponentially dependent on voltage, with beta more steeply voltage dependent, changing e-fold for 9 mV compared with 18 mV for an e-fold change in alpha. Over all [K+]o studied, the voltage dependence of alpha and beta shifted along with EK, as is characteristic of IR channels in other cells. The steady-state voltage dependence of the gating process was well described by a Boltzmann function. The half-activation potential was on average approximately 7 mV negative to the observed reversal potential in all [K+]o regardless of [Mg2+]i. The activation curve was somewhat steeper when Mg-free pipette solutions were used (slope factor, 4.3 mV) than when pipettes contained 1.9 mM Mg2+ (5.2 mV). The simplest interpretation of these data is that IR channels in bovine pulmonary artery endothelial cells have an intrinsic gating mechanism that is not due to Mg block.

scholarly journals Effects of external Rb+ on inward rectifier K+ channels of bovine pulmonary artery endothelial cells.

1994 ◽  
Vol 103 (4) ◽  
pp. 519-548 ◽  
Author(s):  
M R Silver ◽  
M S Shapiro ◽  
T E DeCoursey
Keyword(s):  
Endothelial Cells ◽  
Steady State ◽  
Pulmonary Artery ◽  
Voltage Dependence ◽  
Reversal Potential ◽  
Inward Rectifier ◽  
Partial Substitution ◽  
Pipette Solution ◽  
K Channels ◽  
Voltage Dependent

Inward rectifier (IR) K+ channels of bovine pulmonary artery endothelial cells were studied using the whole-cell, cell-attached, and outside-out patch-clamp configurations. The effects of Rb+ on the voltage dependence and kinetics of IR gating were explored, with [Rb+]o + [K+]o = 160 mM. Partial substitution of Rb+ for K+ resulted in voltage-dependent reduction of inward currents, consistent with Rb+ being a weakly permeant blocker of the IR. In cells studied with a K(+)-free pipette solution, external Rb+ reduced inward IR currents to a similar extent at large negative potentials but block at more positive potentials was enhanced. In outside-out patches, the single-channel i-V relationship was approximately linear in symmetrical K+, but rectified strongly outwardly in high [Rb+]o due to a reduced conductance for inward current. The permeability of Rb+ based on reversal potential, Vrev, was 0.45 that of K+, whereas the Rb+ conductance was much lower, 0.034 that of K+, measured at Vrev-80 mV. The steady state voltage-dependence of IR gating was determined in Rb(+)-containing solutions by applying variable prepulses, followed by a test pulse to a potential at which outward current deactivation was observed. As [Rb+]o was increased, the half-activation potential, V1/2, changed less than Vrev. In high [K+]o solutions V1/2 was Vrev-6 mV, while in high [Rb+]o V1/2 was Vrev + 7 mV. This behavior contrasts with the classical parallel shift of V1/2 with Vrev in K+ solutions. Steady state IR gating was less steeply voltage-dependent in high [Rb+]o than in K+ solutions, with Boltzmann slope factors of 6.4 and 4.4 mV, respectively. Rb+ decreased (slowed) both activation and deactivation rate constants defined at V1/2, and decreased the steepness of the voltage dependence of the activation rate constant by 42%. Deactivation of IR channels in outside-out patches was also slowed by Rb+. In summary, Rb+ can replace K+ in setting the voltage-dependence of IR gating, but in doing so alters the kinetics.

scholarly journals Gating characteristics of a steeply voltage-dependent gap junction channel in rat Schwann cells.

1993 ◽  
Vol 102 (5) ◽  
pp. 925-946 ◽  
Author(s):  
M Chanson ◽  
K J Chandross ◽  
M B Rook ◽  
J A Kessler ◽  
D C Spray
Keyword(s):  
Steady State ◽  
Gap Junction ◽  
Schwann Cells ◽  
Voltage Dependence ◽  
Single Channel ◽  
Neonatal Rat ◽  
Order Kinetic ◽  
Patch Clamp Technique ◽  
Gap Junctional ◽  
Closing Rate

The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, Gss was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary junctional currents was detected corresponding to an unitary channel conductance of approximately 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic Gss voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states.

scholarly journals Mechanism of the Voltage Sensitivity of IRK1 Inward-rectifier K+ Channel Block by the Polyamine Spermine

2005 ◽  
Vol 125 (4) ◽  
pp. 413-426 ◽  
Author(s):  
Hyeon-Gyu Shin ◽  
Zhe Lu
Keyword(s):  
Steady State ◽  
Voltage Dependence ◽  
Ion Selectivity ◽  
Inward Rectifier ◽  
Selectivity Filter ◽  
K Channel ◽  
Voltage Sensitivity ◽  
Channel Block ◽  
Head Groups ◽  
Voltage Dependent

IRK1 (Kir2.1) inward-rectifier K+ channels exhibit exceedingly steep rectification, which reflects strong voltage dependence of channel block by intracellular cations such as the polyamine spermine. On the basis of studies of IRK1 block by various amine blockers, it was proposed that the observed voltage dependence (valence ∼5) of IRK1 block by spermine results primarily from K+ ions, not spermine itself, traversing the transmembrane electrical field that drops mostly across the narrow ion selectivity filter, as spermine and K+ ions displace one another during channel block and unblock. If indeed spermine itself only rarely penetrates deep into the ion selectivity filter, then a long blocker with head groups much wider than the selectivity filter should exhibit comparably strong voltage dependence. We confirm here that channel block by two molecules of comparable length, decane-bis-trimethylammonium (bis-QAC10) and spermine, exhibit practically identical overall voltage dependence even though the head groups of the former are much wider (∼6 Å) than the ion selectivity filter (∼3 Å). For both blockers, the overall equilibrium dissociation constant differs from the ratio of apparent rate constants of channel unblock and block. Also, although steady-state IRK1 block by both cations is strongly voltage dependent, their apparent channel-blocking rate constant exhibits minimal voltage dependence, which suggests that the pore becomes blocked as soon as the blocker encounters the innermost K+ ion. These findings strongly suggest the existence of at least two (potentially identifiable) sequentially related blocked states with increasing numbers of K+ ions displaced. Consequently, the steady-state voltage dependence of IRK1 block by spermine or bis-QAC10 should increase with membrane depolarization, a prediction indeed observed. Further kinetic analysis identifies two blocked states, and shows that most of the observed steady-state voltage dependence is associated with the transition between blocked states, consistent with the view that the mutual displacement of blocker and K+ ions must occur mainly as the blocker travels along the long inner pore.

Block of inwardly rectifying K+ currents by extracellular Mg2+ and Ba2+ in bovine pulmonary artery endothelial cells

2000 ◽  
Vol 78 (9) ◽  
pp. 751-756 ◽  
Author(s):  
Yuk Man Leung ◽  
Chiu Yin Kwan ◽  
Edwin E Daniel
Keyword(s):  
Endothelial Cells ◽  
Patch Clamp ◽  
Pulmonary Artery ◽  
Binding Site ◽  
Patch Clamp Technique ◽  
Aortic Endothelial Cells ◽  
Whole Cell Patch Clamp ◽  
Deep Site ◽  
Low Sensitivity ◽  
Inwardly Rectifying

Using whole-cell patch clamp technique, we investigated the blocking effects of extracellular Ba2+ and Mg2+ on the inwardly rectifying K+ (KIR) currents of bovine pulmonary artery endothelial cells (BPAEC). The BPAEC KIR channel has recently been identified as Kir2.1 of the Kir2.0 subfamily. Block of KIR currents by Mg2+ (3-30 mM) was instantaneous, and increased with hyperpolarization slightly (Kd at -160 and 0 mV was 9.5 and 23.2 mM, respectively). The apparent fractional electrical distance (δ) of the Mg2+ binding site is calculated to be 0.07 from the outer mouth of the channel pore. Ba2+ (0.3-10 µM) time-dependently blocked the KIR currents with a much higher potency and stronger voltage-dependence (Kd at -160 and 0 mV was 1.0 and 41.6 µM, respectively). The Ba2+ binding site had a δ value of 0.34. Our data suggest that Mg2+ binds to a very superficial site of the KIR channel, while Ba2+ binds to a much deeper site, sensing much more of the membrane electric field. Thus, the BPAEC Kir2.1 appears to be pharmacologically different from the Kir2.1 reported before in bovine aortic endothelial cells (BAEC), which has 2 sites for Mg2+ block (a deep site in addition to a shallow one), and a superficial and low-sensitivity site for Ba2+ block.Key words: inwardly rectifying K+ channel, patch clamp, Ba2+, Mg2+, endothelial cells.

Effects of oxidant stress on steady-state background currents in isolated ventricular myocytes

1991 ◽  
Vol 261 (5) ◽  
pp. H1358-H1365 ◽  
Author(s):  
H. Matsuura ◽  
M. J. Shattock
Keyword(s):  
Steady State ◽  
Action Potential ◽  
Rose Bengal ◽  
Oxidant Stress ◽  
Reversal Potential ◽  
Outward Current ◽  
Membrane Conductance ◽  
Inward Rectifier ◽  
Ventricular Cells ◽  
Voltage Axis

Free radicals and oxidant stress have previously been shown to induce depolarization, transient action potential prolongation, and automaticity. We have investigated the ionic basis of these electrophysiological changes in isolated rabbit ventricular cells. Oxidant stress was generated by the photoactivation of rose bengal, and, in current-clamp experiments, the effects of oxidant stress on the action potential were confirmed. In voltage-clamp studies, oxidant stress decreased both inward and outward current through the inward-rectifier potassium channel, and the slope conductance (measured at the voltage-axis intercept near the resting membrane potential) was decreased from 40 +/- 8 to 25 +/- 6 nS (n = 6). Transient inward currents were induced on repolarization after a depolarizing clamp step, suggesting that the cells were calcium overloaded. In addition, oxidant stress activated a steady-state membrane conductance that showed a slight outward-going rectification and a reversal potential of approximately 0 mV. Evidence is presented to indicate that this reflects an increase in the conductance of the calcium-activated nonselective cation channel. The slope conductance of this calcium-activated channel (measured at the voltage-axis intercept) increased with prolonged exposure to oxidant stress (from 0.5 to 12 nS after 7 min), indicating that the intracellular free calcium increased gradually during the maintained application of rose bengal. These results suggest that oxidant stress depolarizes the cell membrane by reducing the inward-rectifier potassium current and by activating a calcium-activated membrane conductance. Both factors may contribute to the oxidant stress-induced changes in action potential duration and automaticity.

scholarly journals Characterization of the inward-rectifying potassium current in cat ventricular myocytes.

1988 ◽  
Vol 91 (4) ◽  
pp. 593-615 ◽  
Author(s):  
R D Harvey ◽  
R E Ten Eick
Keyword(s):  
Steady State ◽  
Time Course ◽  
Voltage Dependence ◽  
Ventricular Myocytes ◽  
Reversal Potential ◽  
Inward Current ◽  
Steady State Current ◽  
Voltage Dependent ◽  
K Current ◽  
K Concentration

Whole-cell membrane currents were measured in isolated cat ventricular myocytes using a suction-electrode voltage-clamp technique. An inward-rectifying current was identified that exhibited a time-dependent activation. The peak current appeared to have a linear voltage dependence at membrane potentials negative to the reversal potential. Inward current was sensitive to K channel blockers. In addition, varying the extracellular K+ concentration caused changes in the reversal potential and slope conductance expected for a K+ current. The voltage dependence of the chord conductance exhibited a sigmoidal relationship, increasing at more negative membrane potentials. Increasing the extracellular K+ concentration increased the maximal level of conductance and caused a shift in the relationship that was directly proportional to the change in reversal potential. Activation of the current followed a monoexponential time course, and the time constant of activation exhibited a monoexponential dependence on membrane potential. Increasing the extracellular K+ concentration caused a shift of this relationship that was directly proportional to the change in reversal potential. Inactivation of inward current became evident at more negative potentials, resulting in a negative slope region of the steady state current-voltage relationship between -140 and -180 mV. Steady state inactivation exhibited a sigmoidal voltage dependence, and recovery from inactivation followed a monoexponential time course. Removing extracellular Na+ caused a decrease in the slope of the steady state current-voltage relationship at potentials negative to -140 mV, as well as a decrease of the conductance of inward current. It was concluded that this current was IK1, the inward-rectifying K+ current found in multicellular cardiac preparations. The K+ and voltage sensitivity of IK1 activation resembled that found for the inward-rectifying K+ currents in frog skeletal muscle and various egg cell preparations. Inactivation of IK1 in isolated ventricular myocytes was viewed as being the result of two processes: the first involves a voltage-dependent change in conductance; the second involves depletion of K+ from extracellular spaces. The voltage-dependent component of inactivation was associated with the presence of extracellular Na+.

scholarly journals Hyperpolarization-Activated, Mixed-Cation Current (I h) in Octopus Cells of the Mammalian Cochlear Nucleus

2000 ◽  
Vol 84 (2) ◽  
pp. 806-817 ◽  
Author(s):  
Ramazan Bal ◽  
Donata Oertel
Keyword(s):  
Cochlear Nucleus ◽  
Voltage Dependence ◽  
Reversal Potential ◽  
Nerve Fibers ◽  
Resting Potential ◽  
Extracellular Potassium ◽  
Dibutyryl Camp ◽  
Cation Current ◽  
Mixed Cation ◽  
Boltzmann Function

Octopus cells in the posteroventral cochlear nucleus of mammals detect the coincidence of synchronous firing in populations of auditory nerve fibers and convey the timing of that coincidence with great temporal precision. Earlier recordings in current clamp have shown that two conductances contribute to the low input resistance and therefore to the ability of octopus cells to encode timing precisely, a low-threshold K+ conductance and a hyperpolarization-activated mixed-cation conductance, g h. The present experiments describe the properties of g h in octopus cells as they are revealed under voltage clamp with whole-cell, patch recordings. The hyperpolarization-activated current, I h, was blocked by extracellular Cs+ (5 mM) and 4-( N-ethyl- N-phenylamino)-1,2-dimethyl-6-(methylamino) pyridinium chloride (50–100 nM) but not by extracellular Ba2+ (2 mM). The reversal potential for I h in octopus cells under normal physiological conditions was −38 mV. Increasing the extracellular potassium concentration from 3 to 12 mM shifted the reversal potential to −26 mV; lowering extracellular sodium concentration from 138 to 10 mM shifted the reversal potential to −77 mV. These pharmacological and ion substitution experiments show that I h in octopus cells is a mixed-cation current that resembles I h in other neurons and in heart muscle cells. Under control conditions when cells were perfused intracellularly with ATP and GTP, I h had an activation threshold between about −35 to −40 mV and became fully activated at −110 mV. The maximum conductance associated with hyperpolarizing voltage steps to −112 mV ranged from 87 to 212 nS [150 ± 30 (SD) nS, n = 36]. The voltage dependence of g h obtained from peak tail currents is fit by a Boltzmann function with a half-activation potential of −65 ± 3 mV and a slope factor of 7.7 ± 0.7. This relationship reveals that g h was activated 41% at the mean resting potential of octopus cells, −62 mV, and that at rest I h contributes a steady inward current of between 0.9 and 2.1 nA. The voltage dependence of g h was unaffected by the extracellular application of dibutyryl cAMP but was shifted in hyperpolarizing direction, independent of the presence or absence of dibutyryl cAMP, by the removal of intracellular ATP and GTP.

Heterogeneous Voltage Dependence of Inward Rectifier Currents in Spiral Ganglion Neurons

1997 ◽  
Vol 78 (6) ◽  
pp. 3019-3027 ◽  
Author(s):  
Zun-Li Mo ◽  
Robin L. Davis
Keyword(s):  
Voltage Clamp ◽  
Voltage Dependence ◽  
Spiral Ganglion ◽  
Reversal Potential ◽  
Cell Types ◽  
Inward Rectifier ◽  
Inward Rectification ◽  
Spiral Ganglion Neurons ◽  
Current Activation ◽  
Ganglion Neurons

Mo, Zun-Li and Robin L. Davis. Heterogeneous voltage dependence of inward rectifier currents in spiral ganglion neurons. J. Neurophysiol. 78: 3019–3027, 1997. Inward rectification was characterized in neonatal spiral ganglion neurons maintained in tissue culture. Whole cell current and voltage-clamp techniques were used to show that the hyperpolarization-activated cationic ( I h) current underlies most or all of the inward rectification demonstrated in these neurons. The average reversal potential (−41.3 mV) and cesium sensitivity were typical of that found in other neurons and cell types. What was unique about the hyperpolarization-activated currents, however, was that the half-maximal voltages ( V 1/2) and slope factors ( k) that characterized I h current activation were graded from neuron to neuron. Voltage-clamp recordings made with standard bath and pipette solutions revealed V 1/2 values that ranged from −78.1 to −122.1 mV, with slope factors from 7.6 to 13.1. These gradations in the voltage-dependent features of the I h current did not result from variability in the recording conditions because independently measured Na+ current-to-voltage relationships were found to be uniform (peak current at −20 mV). Moreover, the range and average V 1/2 and slope values could be altered with activators [8-(4-chlorophenylthio) adenosine 3′,5′-cyclic monophosphate in combination with okadaic acid] or inhibitors { N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide}of protein indicating that I h current heterogeneity most likely resulted from differential phosphorylation.

Two components of calcium currents in the soma of photoreceptors of Hermissenda

1994 ◽  
Vol 72 (3) ◽  
pp. 1327-1336 ◽  
Author(s):  
E. N. Yamoah ◽  
T. Crow
Keyword(s):  
Steady State ◽  
Calcium Currents ◽  
Patch Clamp Technique ◽  
Potential Oscillations ◽  
Generator Potential ◽  
Time To Peak ◽  
Whole Cell Patch Clamp ◽  
Boltzmann Function ◽  
Delayed Time ◽  
Membrane Potential Oscillations

1. The proposed mechanism of cellular plasticity underlying classical conditioning of Hermissenda involves Ca2+ influx through voltage-activated channels. This influx triggers several molecular cascades and leads to the phosphorylation of K+ channels in identified photoreceptors. We studied Ca2+ currents from isolated photoreceptors of Hermissenda with the whole cell patch-clamp technique. Two distinct Ca2+ currents were identified in isolated photoreceptors on the basis of differences in their voltage dependence, kinetics, and pharmacology. 2. One Ca2+ current was transient (ICa(t)), with a fast onset (approximately 5 ms), activated at -50 mV from a holding potential of -90 mV, and peaked at 0 mV. The second Ca2+ current, designated as sustained (ICa(s)), exhibited a delayed time-to-peak, activated at -30 mV, and reached maximum at 30 mV. 3. Steady-state activation curves for both currents were generated from normalized currents and fitted with the Boltzmann function; estimates of half-activation voltages for ICa(t) were -38.8 +/- 6.7 mV (mean +/- SD; n = 9) and 3.2 +/- 8.2 mV for ICa(s) (n = 11) with maximum slopes of 8.9 +/- 1.6 mV (n = 9) and 11.0 +/- 2.4 mV (n = 11). 4. The inactivation of ICa(s) was slow (time constants > 3 s) whereas ICa(t) inactivated rapidly (time constant of inactivation at various voltages; 75-600 ms). 5. Ni2+ (0.8 mM), Gd3+ (0.5 mM), and amiloride (10 microM) produced a reversible block of ICa(t) without affecting ICa(s). omega-Conotoxin GVIA (10 nM) irreversibly blocked ICa(s) whereas nitrendipine (20 microM) produced a reversible block. 6. ICa(t) may be responsible for steady-state membrane potential oscillations. ICa(s) may contribute to the maintenance of the amplitude of the plateau phase of the generator potential.

scholarly journals Aminophylline affects the cardiac rat inward rectifier potassium current in a dual way

EP Europace ◽  
2021 ◽  
Vol 23 (Supplement_3) ◽  
Author(s):  
NJD Ramalho ◽  
O Svecova ◽  
R Kula ◽  
M Simurdova ◽  
J Simurda ◽  
...  
Keyword(s):  
Steady State ◽  
Potassium Current ◽  
Ventricular Myocytes ◽  
Inward Rectifier ◽  
Public Institution ◽  
Patch Clamp Technique ◽  
Rat Ventricular Myocytes ◽  
Funding Sources ◽  
Inward Rectifier Potassium Current ◽  
Isolated Rat

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Ministry of Education, Youth and Sports of the Czech Republic Introduction Aminophylline, a bronchodilator used in clinical practice to treat namely severe astma attacks, often induces atrial fibrillation in patients. Modifications of the inward rectifier potassium current IK1 are known to play a role in the genesis of fibrillation. Purpose We aimed to investigate the effect of aminophylline at clinically-relevant concentrations between 3 and 100 µM on IK1 in isolated rat ventricular myocytes. Methods Experiments were performed by the whole cell patch clamp technique on enzymatically isolated rat right ventricular myocytes at room temperature. IK1 was measured as the current sensitive to 100 µM Ba2+. Results We observed a dual steady-state effect of aminophylline at most of the applied concentrations. Either inhibition or activation was apparent in individual cells during application of aminophylline at a given concentration. The smaller was magnitude of the control IK1, the more likely was activation of the current in the presence of aminophylline and vice versa (tested at 10 and 30 µM). The effect was voltage-independent and fully reversible during the subsequent wash-out. The mean aminophylline effect was inhibitory at all concentrations (10, 15, 4, and 23%-inhibition at -50 mV at 3, 10, 30, and 100 µM, respectively). Using a modified version of the population model of IK1 channels that we published before, the dual effect can be explained by interaction of aminophylline with two channel populations in a different way, the first one being inhibited and the second one being activated by the drug. Considering various fractions of these two channel populations in individual cells, varying effects observed in the measured cells can be simulated. Conclusions Aminophylline at clinically-relevant concentrations affects IK1 in rat ventricular myocytes in a dual way, showing both the steady-state activation and inhibition in various cells, even at the same concentration. It may be related to a different effect of the drug on various Kir2.x subunits forming the heterotetrameric IK1 channels present at the cell membrane of a single cell.

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