scholarly journals The mechanism of inward rectification of potassium channels: "long-pore plugging" by cytoplasmic polyamines.

1995 ◽  
Vol 106 (5) ◽  
pp. 923-955 ◽  
Author(s):  
A N Lopatin ◽  
E N Makhina ◽  
C G Nichols
Keyword(s):  
Steady State ◽  
Outward Current ◽  
Inward Rectifier ◽  
Time Dependent ◽  
Inward Rectification ◽  
Voltage Sensitivity ◽  
Intact Cells ◽  
Time Constants ◽  
Steady State Current ◽  
Activation Phase

The mechanism of inward rectification was examined in cell-attached and inside-out membrane patches from Xenopus oocytes expressing the cloned strong inward rectifier HRK1. Little or no outward current was measured in cell-attached patches. Inward currents reach their maximal value in two steps: an instantaneous phase followed by a time-dependent "activation" phase, requiring at least two exponentials to fit the time-dependent phase. After an activating pulse, the quasi-steady state current-voltage (I-V) relationship could be fit with a single Boltzmann equation (apparent gating charge, Z = 2.0 +/- 0.1, n = 3). Strong rectification and time-dependent activation were initially maintained after patch excision into high [K+] (K-INT) solution containing 1 mM EDTA, but disappeared gradually, until only a partial, slow inactivation of outward current remained. Biochemical characterization (Lopatin, A. N., E. N. Makhina, and C. G. Nichols, 1994. Nature. 372:366-396.) suggests that the active factors are naturally occurring polyamines (putrescine, spermidine, and spermine). Each polyamine causes reversible, steeply voltage-dependent rectification of HRK1 channels. Both the blocking affinity and the voltage sensitivity increased as the charge on the polyamine increased. The sum two Boltzmann functions is required to fit the spermine and spermidine steady state block. Putrescine unblock, like Mg2+ unblock, is almost instantaneous, whereas the spermine and spermidine unblocks are time dependent. Spermine and spermidine unblocks (current activation) can each be fit with single exponential functions. Time constants of unblock change e-fold every 15.0 +/- 0.7 mV (n = 3) and 33.3 +/- 6.4 mV (n = 5) for spermine and spermidine, respectively, matching the voltage sensitivity of the two time constants required to fit the activation phase in cell-attached patches. It is concluded that inward rectification in intact cells can be entirely accounted for by channel block. Putrescine and Mg2+ ions can account for instantaneous rectification; spermine and spermidine provide a slower rectification corresponding to so-called intrinsic gating of inward rectifier K channels. The structure of spermine and spermidine leads us to suggest a specific model in which the pore of the inward rectifier channel is plugged by polyamines that enter deeply into the pore and bind at sites within the membrane field. We propose a model that takes into account the linear structure of the natural polyamines and electrostatic repulsion between two molecules inside the pore. Experimentally observed instantaneous and steady state rectification of HRK1 channels as well as the time-dependent behavior of HRK1 currents are then well fit with the same set of parameters for all tested voltages and concentrations of spermine and spermidine.

Effects of sevoflurane on inward rectifier K+ current in guinea pig ventricular cardiomyocytes

1997 ◽  
Vol 273 (1) ◽  
pp. H324-H332 ◽  
Author(s):  
A. Stadnicka ◽  
Z. J. Bosnjak ◽  
J. P. Kampine ◽  
W. M. Kwok
Keyword(s):  
Steady State ◽  
Guinea Pig ◽  
Outward Current ◽  
Inward Rectifier ◽  
Equilibrium Potential ◽  
Ventricular Cardiomyocytes ◽  
Steady State Current ◽  
Voltage Dependent ◽  
Current Activation ◽  
Extracellular Side

The effects of sevoflurane on the inward rectifier potassium current (IKIR) were examined in guinea pig ventricular cardiomyocytes using the whole cell patch-clamp methodology. Sevoflurane had a unique dual effect on the steady-state current amplitude, producing a reversible, concentration- and voltage-dependent block of the inward current at potentials negative to the potassium equilibrium potential (EK) but enhancing the outward current positive to EK. Accordingly, the steady-state conductance negative to EK was reduced by sevoflurane, but conductance positive to EK was increased. The chord conductance-voltage relationship showed depolarizing shifts at 0.7, 1.3, and 1.6 mM sevoflurane. When the myocytes were dialyzed with 10 mM Mg2+, but not with 1.0 mM Mg2+, sevoflurane further slowed current activation kinetics. With 10 mM intracellular Mg2+, the outward current enhancement by sevoflurane and the associated shifts in half-activation potential were abolished. Polyamines abolished all effects of sevoflurane on IKIR. With the use of the Woodhull model for voltage-dependent block, we determined the sevoflurane interaction site with the inward rectifier potassium channel to be at an electrical distance of 0.2 from the extracellular side.

scholarly journals Chloride channels in mast cells: block by DIDS and role in exocytosis.

1994 ◽  
Vol 104 (6) ◽  
pp. 1099-1111 ◽  
Author(s):  
J Dietrich ◽  
M Lindau
Keyword(s):  
Steady State ◽  
Mast Cells ◽  
Chloride Transport ◽  
Chloride Concentration ◽  
Outward Current ◽  
Chloride Current ◽  
Hill Coefficient ◽  
Disulfonic Acid ◽  
Intact Cells ◽  
Steady State Current

In rat peritoneal mast cells, we have investigated the influence of the chloride transport blocker 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) and the extracellular chloride concentration on the chloride current induced by intracellular application of cyclic AMP (cAMP) and on hexosaminidase secretion from intact cells stimulated with compound 48/80. The inhibition of the Cl-current by extracellular DIDS is voltage and time dependent. Upon depolarization from -10 to +70 mV, the outward current diminishes with millisecond kinetics. The size of the steady state current and the time constant of the decrease both decrease with increasing DIDS concentrations. The steady state current at +70 mV is blocked by DIDS with an IC50 of 2.3 microM. The number of open channels at -10 mV is reduced with an IC50 of 22 microM. The electrophysiological and pharmacological properties of this current are most similar to those of the Cl- current in T lymphocytes activated by osmotic stress (Lewis, R. S., P. E. Ross, and M. D. Cahalan. 1993. Journal of General Physiology. 101:801-826). Extracellular DIDS also inhibits exocytosis. At optimal stimulation with 10 micrograms/ml compound 48/80 secretion is inhibited with an IC50 = 50 microM and a Hill coefficient n = 10. At half optimal stimulation with 1 microgram/ml inhibition occurs with an IC50 = 10 microM and n = 1. Substitution of extracellular chloride by glutamate has only very small effects on secretion stimulated with 10 micrograms/ml compound 48/80. We conclude that activation of the chloride current in mast cells is not essential for stimulation of exocytosis but may enhance secretion at suboptimal stimulation. Alternatively, the channel may play a role in volume regulation following degranulation.

scholarly journals Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium.

1994 ◽  
Vol 103 (4) ◽  
pp. 583-604 ◽  
Author(s):  
V Urbach ◽  
E van Kerkhove ◽  
B J Harvey
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
Ringer Solution ◽  
Inward Rectifier ◽  
Inward Rectification ◽  
K Channel ◽  
Intact Cells ◽  
Principal Cells ◽  
Kir Channel ◽  
Current Voltage

Inward-rectifier K channel: using macroscopic voltage clamp and single-channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current-voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell-attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. Conclusions: under physiological ionic gradients, a 15-pS inward-rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.

Voltage-clamp analysis of the ionic conductances in a leech neuron with a purely calcium-dependent action potential

1987 ◽  
Vol 58 (6) ◽  
pp. 1468-1484 ◽  
Author(s):  
J. Johansen ◽  
J. Yang ◽  
A. L. Kleinhaus
Keyword(s):  
Steady State ◽  
Action Potential ◽  
Time Course ◽  
Outward Current ◽  
Time Constants ◽  
Exponential Time ◽  
Calcium Dependent ◽  
Voltage Dependent ◽  
Ca Currents ◽  
Single Exponential

1. The purely calcium-dependent action potential of the anterior lateral giant (ALG) cell in the leech Haementeria was examined under voltage clamp. 2. Analysis with ion substitutions showed that the ALG cell action potential is generated by only two time- and voltage-dependent conductance systems, an inward Ca-dependent current (ICa) and an outward Ca-dependent K current IK(Ca). 3. The kinetic properties of the inward current were examined both in Cs-loaded neurons with Ca as the current carrier as well as in Ba-containing Ringer solutions with Ba as the current carrier, since Ba effectively blocked all time- and voltage-dependent outward current. 4. During a maintained depolarization, Ba and Ca currents activated with a time constant tau m, they then inactivated with the decay following a single exponential time course with a time constant tau h. The time constants for decay of both Ba and Ca currents were comparable, suggesting that the mechanism of inactivation of ICa in the ALG cell is largely voltage dependent. In the range of potentials from 5 to 45 mV, tau m varied from 8 to 2 ms and tau h varied from 250 to 125 ms. 5. The activation of currents carried by Ba, after correction for inactivation, could be described reasonably well by the expression I'Ba = I'Ba(infinity) [1--exp(-t/tau m)]. 6. The steady-state activation of the Ba-conductance mBa(infinity) increased sigmoidally with voltage and was approximated by the equation mBa(infinity) = (1 + exp[(Vh-6)/3])-1. The steady-state inactivation hBa(infinity) varied with holding potential and could be described by the equation hBa(infinity) = [1 + exp(Vh + 10/7)]-1. Recovery from inactivation of IBa was best described by the sum of two exponential time courses with time constants of 300 ms and 1.75 s, respectively. 7. The outward current IK(Ca) developed very slowly (0.5–1 s to half-maximal amplitude) and did not inactivate during a 20-s depolarizing command pulse. Tail current decay of IK(Ca) followed a single exponential time course with voltage-dependent time constants of between 360 and 960 ms. The steady-state activation n infinity of IK(Ca) increased sigmoidally with depolarization as described by the equation n infinity = [1 + exp(Vh-13.5)/-8)]-1. 8. The reversal potentials of IK(Ca) tail currents were close to the expected equilibrium potential for potassium and they varied linearly with log [K]o with a slope of 51 mV. These results suggest a high selectivity of the conductance for K ions.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine reduces inward rectification on thymus-derived macrophage cells in culture

1987 ◽  
Vol 65 (3) ◽  
pp. 348-351 ◽  
Author(s):  
F. Moody-Corbett ◽  
P. Brehm
Keyword(s):  
Outward Current ◽  
Inward Rectifier ◽  
Cholinergic Innervation ◽  
Equilibrium Potential ◽  
Inward Rectification ◽  
Inward Current ◽  
Carbon Particles ◽  
Macrophage Cells ◽  
Anomalous Rectifier ◽  
Cell Current

Cultures prepared from dissociated rat thymus were examined 1–2 weeks after plating. Macrophage cells were identified by their adherence, morphological appearance, and ability to phagocytize carbon particles or heat-inactivated Staphylococcus aureus. Whole cell current recordings from macrophage cells revealed an inward current at potentials more negative than the equilibrium potential for potassium and an outward current at potentials more positive than −40 mV in normal recording solution. Acetylcholine or muscarine caused a reduction in inward current but did not alter the outward current. The inward current and acetylcholine effect were seen at less negative potentials by decreasing the potassium equilibrium potential and both were blocked by the addition of cesium to the external recording solution. These results indicated that the inward current was mediated by potassium through the inward or anomalous rectifier. Physiologically, the action of acetylcholine on the inward rectifier of these macrophage cells may be mediated by cholinergic innervation of the thymus.

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.

scholarly journals Na Self Inhibition of Human Epithelial Na Channel

2002 ◽  
Vol 120 (2) ◽  
pp. 133-145 ◽  
Author(s):  
Ahmed Chraïbi ◽  
Jean-Daniel Horisberger
Keyword(s):  
Steady State ◽  
Reversal Potential ◽  
Outward Current ◽  
Inactivation Rate ◽  
Na Channel ◽  
Temperature Dependent ◽  
Open Probability ◽  
Inward Current ◽  
Steady State Current ◽  
Epithelial Na Channel

The regulation of the open probability of the epithelial Na+ channel (ENaC) by the extracellular concentration of Na+, a phenomenon called “Na+ self inhibition,” has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na+ self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na+ concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q10act = 1.5) activation rate and a strongly temperature-dependant (Q10inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na+ self inhibition depended only on the extracellular Na+ concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na+ as well as a reduction of Na+ outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na+ self inhibition is an intrinsic property of sodium channels resulting from the expression of the α, β, and γ subunits of human ENaC in Xenopus oocyte. The extracellular Na+-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases.

Four different components contribute to outward current in rat ventricular myocytes

1999 ◽  
Vol 277 (1) ◽  
pp. H107-H118 ◽  
Author(s):  
Herbert M. Himmel ◽  
Erich Wettwer ◽  
Qi Li ◽  
Ursula Ravens
Keyword(s):  
Steady State ◽  
Ventricular Myocytes ◽  
Outward Current ◽  
Cell Voltage ◽  
Differential Sensitivity ◽  
Resting Potential ◽  
Residual Fraction ◽  
Voltage Range ◽  
Rat Ventricular Myocytes ◽  
Steady State Current

In rat ventricle, two Ca2+-insensitive components of K+ current have been distinguished kinetically and pharmacologically, the transient, 4-aminopyridine (4-AP)-sensitive I to and the sustained, tetraethylammonium (TEA)-sensitive I K. However, a much greater diversity of depolarization-activated K+ channels has been reported on the level of mRNA and protein. In the search for electrophysiological evidence of further current components, the whole cell voltage-clamp technique was used to analyze steady-state inactivation of outward currents by conditioning potentials in a wide voltage range. Peak ( I peak) and late ( I late) currents during the test pulse were analyzed by Boltzmann curve fitting, producing three fractions each. Fractions a and b had different potentials of half-maximum inactivation ( V 0.5); the third residual fraction, r, did not inactivate. Fractions a for I peak and I late had similar relative amplitudes and V 0.5 values, whereas size and V 0.5 of fractions b differed significantly between I peak and I late. Only b of I peak was transient, suggesting a relation with I to, whereas a, b, and r of I late appeared to be three different sustained currents. Therefore, four individual outward current components were distinguished: I to( b of I peak), I K( a), the steady-state current I ss( r), and the novel current I Kx( b of I late). This was further supported by differential sensitivity to TEA, 4-AP, clofilium, quinidine, dendrotoxin, heteropodatoxin, and hanatoxin. With the exception of I to, none of the currents exhibited a marked transmural gradient. Availability of I K was low at resting potential; nevertheless, I K contributed to action potential shortening in hyperpolarized subendocardial myocytes. In conclusion, on the basis of electrophysiological and pharmacological evidence, at least four components contribute to outward current in rat ventricular myocytes.

Long-Term Effects of Prior Heat Shock on Neuronal Potassium Currents Recorded in a Novel Insect Ganglion Slice Preparation

1999 ◽  
Vol 81 (2) ◽  
pp. 795-802 ◽  
Author(s):  
J. M. Ramirez ◽  
F. P. Elsen ◽  
R. M. Robertson
Keyword(s):  
Steady State ◽  
Heat Shock ◽  
Outward Current ◽  
Potassium Currents ◽  
Respiratory Neurons ◽  
Long Term Effects ◽  
Slice Preparation ◽  
Population Activity ◽  
Steady State Current

Long-term effects of prior heat shock on neuronal potassium currents recorded in a novel insect ganglion slice preparation. Brief exposure to high temperatures (heat shock) induces long-lasting adaptive changes in the molecular biology of protein interactions and behavior of poikilotherms. However, little is known about heat shock effects on neuronal properties. To investigate how heat shock affects neuronal properties we developed an insect ganglion slice from locusts. The functional integrity of neuronal circuits in slices was demonstrated by recordings from rhythmically active respiratory neurons and by the ability to induce rhythmic population activity with octopamine. Under these “functional” in vitro conditions we recorded outward potassium currents from neurons of the ventral midline of the A1 metathoracic neuromere. In control neurons, voltage steps to 40 mV from a holding potential of −60 mV evoked in control neurons potassium currents with a peak current of 10.0 ± 2.5 nA and a large steady state current of 8.5 ± 2.6 nA, which was still activated from a holding potential of −40 mV. After heat shock most of the outward current inactivated rapidly (peak amplitude: 8.4 ± 2.4 nA; steady state: 3.6 ± 2.0 nA). This current was inactivated at a holding potential of −40 mV. The response to temperature changes was also significantly different. After changing the temperature from 38 to 42°C the amplitude of the peak and steady-state current was significantly lower in neurons obtained from heat-shocked animals than those obtained from controls. Our study indicates that not only heat shock can alter neuronal properties, but also that it is possible to investigate ion currents in insect ganglion slices.

Ammonium interaction with the epithelial sodium channel

2001 ◽  
Vol 281 (3) ◽  
pp. F493-F502 ◽  
Author(s):  
Nazih L. Nakhoul ◽  
Kathleen S. Hering-Smith ◽  
Solange M. Abdulnour-Nakhoul ◽  
L. Lee Hamm
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Sodium Channel ◽  
Outward Current ◽  
Intracellular Acidification ◽  
Positive Potential ◽  
Steady State Current ◽  
Electrode Voltage ◽  
Ion Activities ◽  
Cl Permeability

The purpose of this study was to investigate the direct effect of NH3/NH[Formula: see text] on mouse epithelial Na+ channels (mENaC) expressed in Xenopusoocytes. Two-electrode voltage-clamp and ion-selective microelectrodes were used to measure the Na+ current, intracellular pH (pHi), and ion activities in oocytes expressing mENaC. In oocytes expressing mENaC, removal of external Na+reversibly hyperpolarized membrane potential by 129 ± 5.3 mV in the absence of 20 mM NH4Cl but only by 100 ± 7.8 mV in its presence. Amiloride completely inhibited the changes in membrane potential. In oocytes expressing mENaC, butyrate (20 mM) caused a decrease in pHi (0.43 ± 0.07) similar to the NH4Cl-induced pHi decrease (0.47 ± 0.12). Removal of Na+ in the presence of butyrate caused hyperpolarization that was not significantly different from that in the absence of butyrate at high pHi (in the absence of NH4Cl). Removal of external Na+ resulted in an outward current of 3.7 ± 0.8 μA (at −60 mV). The magnitude of this change in current was only 2.7 ± 0.7 μA when Na+ was removed in the presence of NH4Cl. In oocytes expressing mENaC, NH4Cl also caused a decrease in whole cell conductance at negative potential and an outward current at positive potential. In the presence of amiloride, steady-state current and the change in current caused by removal of Na+ were not different from zero. These results indicate that NH4Cl inhibits Na+ transport when mENaC is expressed in oocytes. The inhibition of voltage changes is not due to intracellular acidification caused by NH4Cl. Permeability and selectivity of ENaC to NH[Formula: see text] may play a role.

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