scholarly journals A rapidly activating and slowly inactivating potassium channel cloned from human heart. Functional analysis after stable mammalian cell culture expression.

1993 ◽  
Vol 101 (4) ◽  
pp. 513-543 ◽  
Author(s):  
D J Snyders ◽  
M M Tamkun ◽  
P B Bennett
Keyword(s):  
Cell Lines ◽  
Time Course ◽  
Reversal Potential ◽  
Delayed Rectifier ◽  
Inactivation Kinetics ◽  
Constant Field ◽  
Slow Inactivation ◽  
Time Constants ◽  
K Concentration ◽  
External K

The electrophysiological properties of HK2 (Kv1.5), a K+ channel cloned from human ventricle, were investigated after stable expression in a mouse Ltk- cell line. Cell lines that expressed HK2 mRNA displayed a current with delayed rectifier properties at 23 degrees C, while sham transfected cell lines showed neither specific HK2 mRNA hybridization nor voltage-activated currents under whole cell conditions. The expression of the HK2 current has been stable for over two years. The dependence of the reversal potential of this current on the external K+ concentration (55 mV/decade) confirmed K+ selectivity, and the tail envelope test was satisfied, indicating expression of a single population of K+ channels. The activation time course was fast and sigmoidal (time constants declined from 10 ms to < 2 ms between 0 and +60 mV). The midpoint and slope factor of the activation curve were Eh = -14 +/- 5 mV and k = 5.9 +/- 0.9 (n = 31), respectively. Slow partial inactivation was observed especially at large depolarizations (20 +/- 2% after 250 ms at +60 mV, n = 32), and was incomplete in 5 s (69 +/- 3%, n = 14). This slow inactivation appeared to be a genuine gating process and not due to K+ accumulation, because it was present regardless of the size of the current and was observed even with 140 mM external K+ concentration. Slow inactivation had a biexponential time course with largely voltage-independent time constants of approximately 240 and 2,700 ms between -10 and +60 mV. The voltage dependence of slow inactivation overlapped with that of activation: Eh = -25 +/- 4 mV and k = 3.7 +/- 0.7 (n = 14). The fully activated current-voltage relationship displayed outward rectification in 4 mM external K+ concentration, but was more linear at higher external K+ concentrations, changes that could be explained in part on the basis of constant field (Goldman-Hodgkin-Katz) rectification. Activation and inactivation kinetics displayed a marked temperature dependence, resulting in faster activation and enhanced inactivation at higher temperature. The current was sensitive to low concentrations of 4-aminopyridine, but relatively insensitive to external TEA and to high concentrations of dendrotoxin. The expressed current did not resemble either the rapid or the slow components of delayed rectification described in guinea pig myocytes. However, this channel has many similarities to the rapidly activating delayed rectifying currents described in adult rat atrial and neonatal canine epicardial myocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Effect of acetylcholine on postjunctional membrane permeability in eel electroplaque.

1977 ◽  
Vol 70 (1) ◽  
pp. 23-36 ◽  
Author(s):  
N L Lassignal ◽  
A R Martin
Keyword(s):  
Field Equation ◽  
Membrane Potential ◽  
Membrane Permeability ◽  
Ionic Composition ◽  
Reversal Potential ◽  
Resting Potential ◽  
Constant Field ◽  
Free Solution ◽  
Positive Direction ◽  
External K

Acetylcholine (ACh) was applied iontophoretically to the innervated face of isolated eel electroplaques while the membrane potential was being recorded intracellularly. At the resting potential (about -85 mV) application of the drug produced depolarizations (ACh potentials) of 20 mV or more which became smaller when the membrane was depolarized and reversed in polarity at about zero membrane potential. The reversal potential shifted in the negative direction when external Na+ was partially replaced by glucosamine. Increasing external K+ caused a shift of reversal potential in the positive direction. It was concluded that ACh increased the permeability of the postjunctional membrane to both ions. Replacement of Cl- by propionate had no effect on the reversal potential. In Na+-free solution containing glucosamine the reversal potential was positive to the resting potential, suggesting that ACh increased the permeability to glucosamine. Addition of Ca++ resulted in a still more positive reversal potential, indicating an increased permeability to Ca++ as well. Analysis of the results indicated that the increases in permeability of the postjunctional membrane to K+, Na+, Ca++, and glucosamine were in the ratios of approximately 1.0:0.9:0.7:0.2, respectively. With these permeability ratios, all of the observed shifts in reversal potential with changes in external ionic composition were predicted accurately by the constant field equation.

scholarly journals Ionic Basis of Tonic Firing in Spinal Substantia Gelatinosa Neurons of Rat

2004 ◽  
Vol 91 (2) ◽  
pp. 646-655 ◽  
Author(s):  
Igor V. Melnick ◽  
Sónia F. A. Santos ◽  
Karolina Szokol ◽  
Péter Szûcs ◽  
Boris V. Safronov
Keyword(s):  
Time Course ◽  
Substantia Gelatinosa ◽  
Resting Potential ◽  
Delayed Rectifier ◽  
Inactivation Kinetics ◽  
Basic Pattern ◽  
Tonic Firing ◽  
Recovery From Inactivation ◽  
Ionic Conductances ◽  
Fast Activation

Ionic conductances underlying excitability in tonically firing neurons (TFNs) from substantia gelatinosa (SG) were studied by the patch-clamp method in rat spinal cord slices. Ca2+-dependent K+ (KCA) conductance sensitive to apamin was found to prolong the interspike intervals and stabilize firing evoked by a sustained membrane depolarization. Suppression of Ca2+ and KCA currents, however, did not abolish the basic pattern of tonic firing, indicating that it was generated by voltage-gated Na+ and K+ currents. Na+ and K+ channels were further analyzed in somatic nucleated patches. Na+ channels exhibited fast activation and inactivation kinetics and followed two-exponential time course of recovery from inactivation. The major K+ current was carried through tetraethylammonium (TEA)-sensitive rapidly activating delayed-rectifier (KDR) channels with a slow inactivation. The TEA-insensitive transient A-type K+ (KA) current was very small in patches and was strongly inactivated at resting potential. Block of KDR rather than KA conductance by 1 mM TEA lowered the frequency and stability of firing. Intracellular staining with biocytin revealed at least three morphological groups of TFNs. Finally, on the basis of present data, we created a model of TFN and showed that Na+ and KDR currents are sufficient to generate a basic pattern of tonic firing. It is concluded that the balanced contribution of all ionic conductances described here is important for generation and modulation of tonic firing in SG neurons.

scholarly journals Energetics of Shaker K channels block by inactivation peptides.

1993 ◽  
Vol 102 (6) ◽  
pp. 977-1003 ◽  
Author(s):  
R D Murrell-Lagnado ◽  
R W Aldrich
Keyword(s):  
Ionic Strength ◽  
Electrostatic Interactions ◽  
Time Course ◽  
Free Peptide ◽  
Terminal Domain ◽  
Voltage Dependent ◽  
Inactivation Process ◽  
K Concentration ◽  
Association Rate Constants ◽  
External K

A synthetic peptide of the NH2-terminal inactivation domain of the ShB channel blocks Shaker channels which have an NH2-terminal deletion and mimics many of the characteristics of the intramolecular inactivation reaction. To investigate the role of electrostatic interactions in both peptide block and the inactivation process we measured the kinetics of block of macroscopic currents recorded from the intact ShB channel, and from ShB delta 6-46 channels in the presence of peptides, at different ionic strengths. The rate of inactivation and the association rate constants (k(on)) for the ShB peptides decreased with increasing ionic strength. k(on) for a more positively charged peptide was more steeply dependent on ionic strength consistent with a simple electrostatic mechanism of enhanced diffusion. This suggests that a rate limiting step in the inactivation process is the diffusion of the NH2-terminal domain towards the pore. The dissociation rates (k(off)) were insensitive to ionic strength. The temperature dependence of k(on) for the ShB peptide was very high, (Q10 = 5.0 +/- 0.58), whereas k(off) was relatively temperature insensitive (Q10 approximately 1.1). The results suggest that at higher temperatures the proportion of time either the peptide or channel spends in the correct conformation for binding is increased. There were two components to the time course of recovery from block by the ShB peptide, indicating two distinct blocked states, one of which has similar kinetics and dependence on external K+ concentration as the inactivated state of ShB. The other is voltage-dependent and at -120 mV is very unstable. Increasing the net charge on the peptide did not increase sensitivity to knock-off by external K+. We propose that the free peptide, having fewer constraints than the tethered NH2-terminal domain binds to a similar site on the channel in at least two different conformations.

5-HT modulation of hyperpolarization-activated inward current and calcium-dependent outward current in a crustacean motor neuron

1992 ◽  
Vol 68 (2) ◽  
pp. 496-508 ◽  
Author(s):  
O. Kiehn ◽  
R. M. Harris-Warrick
Keyword(s):  
Motor Neuron ◽  
Time Course ◽  
Reversal Potential ◽  
Outward Current ◽  
Resting Membrane Potential ◽  
Inward Current ◽  
Calcium Dependent ◽  
Voltage Dependent ◽  
K Concentration ◽  
Conductance Increase

1. Serotonergic modulation of a hyperpolarization-activated inward current, Ih, and a calcium-dependent outward current, Io(Ca), was examined in the dorsal gastric (DG) motor neuron, with the use of intracellular recording techniques in an isolated preparation of the crab stomatogastric ganglion (STG). 2. Hyperpolarization of the membrane from rest with maintained current pulses resulted in a slow time-dependent relaxation back toward rest and a depolarizing overshoot after termination of the current pulse. In voltage clamp, hyperpolarizing commands negative to approximately -70 mV caused a slowly developing inward current, Ih, which showed no inactivation. Repolarization back to the holding potential of -50 mV revealed a slow inward tail current. 3. The reversal potential for Ih was approximately -35 mV. Raising extracellular K+ concentration ([K+]o) from 11 to 22 mM enhanced, whereas decreasing extracellular Na+ concentration ([Na+]o) reduced the amplitude of Ih. These results indicate that Ih in DG is carried by both K+ and Na+ ions. 4. Bath application of serotonin (5-HT; 10 microM) caused a marked increase in the amplitude of Ih through its active voltage ranges. 5. The time course of activation of Ih was well fitted by a single exponential function and strongly voltage dependent. 5-HT increased the rate of activation of Ih. 5-HT also slowed the rate of deactivation of the Ih tail on repolarization to -50 mV. 6. The activation curve for the conductance (Gh) underlying Ih was obtained by analyzing tail currents. 5-HT shifted the half activation for Gh from approximately -105 mV in control to -95 mV, resulting in an increase in the amplitude of Gh active at rest. 7. Two to 4 mM Cs+ abolished Ih, whereas barium (200 microM to 2 mM) had only weak suppressing effects on Ih. Concomitantly, Cs+ also blocked the 5-HT-induced inward current and conductance increase seen at voltages negative to rest. In current clamp, Cs+ caused DG to hyperpolarize 3-4 mV from rest, suggesting that Ih is partially active at rest and contributes to the resting membrane potential. 8. Depolarizing voltage commands from a holding potential of -50 mV resulted in a total outward current (Io) with an initial transient component and a sustained steady-state component. Application of 5-HT reduced both the transient and sustained components of Io. 9. Io was reduced by 10-20 mM tetraethylammonium (TEA), suggesting that it is primarily a K+ current.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane Potentials in the Muscles of the South American Frog, Leptodactylus Ocelatus

1955 ◽  
Vol 32 (3) ◽  
pp. 539-546
Author(s):  
E. J. HARRIS ◽  
H. MARTINS-FERREIRA
Keyword(s):  
Ringer Solution ◽  
Resting Potential ◽  
Phosphate Solution ◽  
Constant Field ◽  
South American ◽  
The South ◽  
American Frog ◽  
K Concentration ◽  
Phosphate Solutions ◽  
External K

1. The resting potential of the fibres of the South American frog Leptodactylus ocelatus has been measured using microelectrodes. 2. In 2 mM. potassium Ringer solution values of 85-103 mV. were obtained. The value for a given muscle depends upon the external K concentration and the relation between resting potential and the concentrations of K, Na and Cl in the medium can be fitted to the constant field solution of the diffusion equation. 3. Use of phosphate solutions in place of chloride solutions led to lower resting potentials at a given K concentration, which is understandable since the contribution of Cl ions to the resting potential is not present in the phosphate solution. 4. The action potential was observed. Its rate of fall was less when the positive overshoot was low than when the overshoot was high. 5. Analyses of muscles and plasma were made.

Characterization of a delayed rectifier potassium current in chicken growth plate chondrocytes

1992 ◽  
Vol 262 (5) ◽  
pp. C1335-C1340 ◽  
Author(s):  
K. B. Walsh ◽  
S. D. Cannon ◽  
R. E. Wuthier
Keyword(s):  
Growth Plate ◽  
Potassium Current ◽  
Reversal Potential ◽  
Delayed Rectifier ◽  
Dependent Manner ◽  
Growth Plate Chondrocytes ◽  
Patch Clamp Technique ◽  
Delayed Rectifier Potassium Current ◽  
Current Activation ◽  
External K

With the use of the whole cell arrangement of the patch-clamp technique, an outward-directed time-dependent potassium current was identified in cultured chicken growth plate chondrocytes. This delayed rectifier potassium current (IK) activated with a sigmoidal time course during voltage steps to potentials positive to -40 mV. The half-maximal voltage required for current activation was determined to be -8 mV. The reversal potential (Erev) for IK, measured using deactivating tail currents, was -72 mV in the presence of 140 mM internal and 5 mM external [K+] solutions. Changes in external [K+] caused Erev to shift in a manner expected for a potassium-selective channel. In addition, increasing external [K+] from 5 to 50 mM caused the slope conductance of the tail currents to increase twofold. The chondrocyte IK was inhibited by the potassium-channel blocker 4-aminopyridine (4-AP) at concentrations of 0.5-4 mM and by the scorpion venom toxin charybdotoxin (CTX; 10 nM) but was unaffected by 10 mM tetraethylammonium (TEA). Addition of 20 microM ZnCl2 reduced IK in a voltage-dependent manner with the greatest inhibition found to occur at potentials near the threshold for current activation. Reduction of IK by ZnCl2 was accompanied by a slowing in the kinetics of IK activation. On the basis of the gating and pharmacological properties of this current, it is suggested that the chondrocyte channel belongs to a superfamily of K+ channels found in bone and immune system cells. The chondrocyte K+ channel may contribute to the unusually high [K+] found in the extracellular fluid of growth plate cartilage.

scholarly journals Slow Inactivation in Shaker K Channels Is Delayed by Intracellular Tetraethylammonium

2008 ◽  
Vol 132 (6) ◽  
pp. 633-650 ◽  
Author(s):  
Vivian González-Pérez ◽  
Alan Neely ◽  
Christian Tapia ◽  
Giovanni González-Gutiérrez ◽  
Gustavo Contreras ◽  
...  
Keyword(s):  
Time Course ◽  
Inactivation Kinetics ◽  
Type I ◽  
Slow Inactivation ◽  
External Application ◽  
K Channels ◽  
Gating Charge ◽  
Channel Opening ◽  
Activation Gate ◽  
Free Membrane

After removal of the fast N-type inactivation gate, voltage-sensitive Shaker (Shaker IR) K channels are still able to inactivate, albeit slowly, upon sustained depolarization. The classical mechanism proposed for the slow inactivation observed in cell-free membrane patches—the so called C inactivation—is a constriction of the external mouth of the channel pore that prevents K+ ion conduction. This constriction is antagonized by the external application of the pore blocker tetraethylammonium (TEA). In contrast to C inactivation, here we show that, when recorded in whole Xenopus oocytes, slow inactivation kinetics in Shaker IR K channels is poorly dependent on external TEA but severely delayed by internal TEA. Based on the antagonism with internally or externally added TEA, we used a two-pulse protocol to show that half of the channels inactivate by way of a gate sensitive to internal TEA. Such gate had a recovery time course in the tens of milliseconds range when the interpulse voltage was −90 mV, whereas C-inactivated channels took several seconds to recover. Internal TEA also reduced gating charge conversion associated to slow inactivation, suggesting that the closing of the internal TEA-sensitive inactivation gate could be associated with a significant amount of charge exchange of this type. We interpreted our data assuming that binding of internal TEA antagonized with U-type inactivation (Klemic, K.G., G.E. Kirsch, and S.W. Jones. 2001. Biophys. J. 81:814–826). Our results are consistent with a direct steric interference of internal TEA with an internally located slow inactivation gate as a “foot in the door” mechanism, implying a significant functional overlap between the gate of the internal TEA-sensitive slow inactivation and the primary activation gate. But, because U-type inactivation is reduced by channel opening, trapping the channel in the open conformation by TEA would also yield to an allosteric delay of slow inactivation. These results provide a framework to explain why constitutively C-inactivated channels exhibit gating charge conversion, and why mutations at the internal exit of the pore, such as those associated to episodic ataxia type I in hKv1.1, cause severe changes in inactivation kinetics.

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+.

Properties of inward calcium current in guinea pig ureteral myocytes

1997 ◽  
Vol 272 (2) ◽  
pp. C543-C549 ◽  
Author(s):  
J. L. Sui ◽  
C. Y. Kao
Keyword(s):  
Action Potentials ◽  
Calcium Current ◽  
Inactivation Kinetics ◽  
Slow Inactivation ◽  
Time Constants ◽  
Channel Inactivation ◽  
Steady State Inactivation ◽  
Window Current ◽  
Kinetics Of ◽  
Ca2 Channels

Ureteral myocytes of guinea pigs have L-type Ca2+ channels (I(Ca)). In 3 mM Ca2+, maximum I(Ca) was 3.38 microA/cm2. Voltage at which conductance is 50% of maximum (V0.5) of I(Ca) was -1.0 mV in 3 mM Ca2+ and +22 mV in 30 mM Ca2+, with slope factors of 8 mV. V0.5 of steady-state inactivation of I(Ca) was -16.2 and +1.1 mV in 3 and 30 mM Ca2+, respectively, with similar slope factors of about -6 mV. A window current reaching 20-25% of the maximum I(Ca) was active between -20 and 0 mV. I(Ca) inactivated very slowly, with time constants of 217.6 and 2,455.9 ms with no voltage dependency. When Ba2+ was used as the charge carrier, the amplitude and inactivation kinetics of the Ba2+ current were similar to those for I(Ca). These results indicate that the ureteral myocyte has little Ca2+-mediated Ca2+ channel inactivation, a feature significantly associated with the slow I(Ca) inactivation. The slow inactivation and the window current are essential for the sustained membrane depolarization during the plateau of ureteral action potentials.

scholarly journals Inward current in single smooth muscle cells of the guinea pig taenia coli.

1989 ◽  
Vol 93 (3) ◽  
pp. 521-550 ◽  
Author(s):  
Y Yamamoto ◽  
S L Hu ◽  
C Y Kao
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Time Course ◽  
Reversal Potential ◽  
Muscle Cells ◽  
Taenia Coli ◽  
Average Cell ◽  
Time Constants ◽  
Inward Current

Using the tight-seal voltage-clamp method, the ionic currents in the enzymatically dispersed single smooth muscle cells of the guinea pig taenia coli have been studied. In a physiological medium containing 3 mM Ca2+, the cells are gently tapering spindles, averaging 201 (length) x 8 microns (largest diameter in center of cell), with a volume of 5 pl. The average cell capacitance is 50 pF, and the specific membrane capacitance 1.15 microF/cm2. The input impedance of the resting cell is 1-2 G omega. Spatially uniform voltage-control prevails after the first 400 microseconds. There is much overlap of the inward and outward currents, but the inward current can be isolated by applying Cs+ internally to block all potassium currents. The inward current is carried by Ca2+. Activation begins at approximately -30 mV, maximum ICa occurs at +10-+20 mV, and the reversal potential is approximately +75 mV. The Ca2+ channel is permeable to Sr2+ and Ba2+, and to Cs+ moving outwards, but not to Na+ moving inwards. Activation and deactivation are very rapid at approximately 33 degrees C, with time-constants of less than 1 ms. Inactivation has a complex time course, resolvable into three exponential components, with average time constants (at 0 mV) of 7, 45, and 400 ms, which are affected differently by voltage. Steady-state inactivation is half-maximal at -30 mV for all components combined, but -36 mV for the fast component and -26 and -23 mV for the other two components. The presence of multiple forms of Ca2+ channel is inferred from the inactivation characteristics, not from activation properties. Recovery of the fast channel occurs with a time-constant of 72 ms (at +10 mV). Ca2+ influx during an action potential can transfer approximately 9 pC of charge, which could elevate intracellular Ca2+ concentration adequately for various physiological functions.

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