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.

Changes in the intracellular free calcium concentration of Aplysia and leech neurones measured with calcium-sensitive microelectrodes

1987 ◽  
Vol 65 (5) ◽  
pp. 934-939 ◽  
Author(s):  
Joachim W. Deitmer ◽  
Roger Eckert ◽  
Wolf-R. Schlue
Keyword(s):  
Calcium Concentration ◽  
Neuronal Membrane ◽  
Free Calcium ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
K Current ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration ◽  
K Concentration ◽  
External K

The intracellular free Ca concentration was measured in invertebrate neurones using single-barrelled and double-barrelled neutral-carrier microelectrodes. The electrodes were calibrated in solutions containing different Ca concentrations between 1 mM and 0.01 μM. The electrode responses were also tested at different ionic strengths and at varying Na concentrations. The electrodes responded with 25–30 mV per 10-fold change in Ca concentration between 1 mM and 1 μM and with 10–25 mV between 1 and 0.1 μM Ca. The intracellular free Ca concentration was measured to be between 0.1 and 0.7 μM in the neurones. The changes of intracellular Ca in identified voltage-clamped neurones of Aplysia californica were recorded during iontophoretic injections of Ca2+ or EGTA. The decrease of intracellular Ca following EGTA injection was correlated with the suppression of the Ca-dependent K current and with the reduction of Ca-induced inactivation of voltage-dependent Ca current. In identified neurones of the leech Hirudo medicinalis a reversible increase of intracellular Ca2+ was recorded after inhibition of the Na–K pump, either by addition of ouabain (0.5 mM) or by lowering the external K concentration (0.2 mM). This rise in intracellular Ca2+ did not occur, and was even reversed, in the absence of external Na, suggesting the existence of Na–Ca exchange across the leech neuronal membrane.

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)

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 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 A Quantitative Description of KcsA Gating I: Macroscopic Currents

2007 ◽  
Vol 130 (5) ◽  
pp. 465-478 ◽  
Author(s):  
Sudha Chakrapani ◽  
Julio F Cordero-Morales ◽  
Eduardo Perozo
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Ph Dependence ◽  
Quantitative Description ◽  
Hill Coefficient ◽  
K Channel ◽  
Open Probability ◽  
Transmembrane Voltage ◽  
Voltage Dependent ◽  
Inactivation Process

The prokaryotic K+ channel KcsA is activated by intracellular protons and its gating is modulated by transmembrane voltage. Typically, KcsA functions have been studied under steady-state conditions, using macroscopic Rb+-flux experiments and single-channel current measurements. These studies have provided limited insights into the gating kinetics of KcsA due to its low open probability, uncertainties in the number of channels in the patch, and a very strong intrinsic kinetic variability. In this work, we have carried out a detailed analysis of KcsA gating under nonstationary conditions by examining the influence of pH and voltage on the activation, deactivation, and slow-inactivation gating events. We find that activation and deactivation gating of KcsA are predominantly modulated by pH without a significant effect of voltage. Activation gating showed sigmoidal pH dependence with a pKa of ∼4.2 and a Hill coefficient of ∼2. In the sustained presence of proton, KcsA undergoes a time-dependent decay of conductance. This inactivation process is pH independent but is modulated by voltage and the nature of permeant ion. Recovery from inactivation occurs via deactivation and also appears to be voltage dependent. We further find that inactivation in KcsA is not entirely a property of the open-conducting channel but can also occur from partially “activated” closed states. The time course of onset and recovery of the inactivation process from these pre-open closed states appears to be different from the open-state inactivation, suggesting the presence of multiple inactivated states with diverse kinetic pathways. This information has been analyzed together with a detailed study of KcsA single-channel behavior (in the accompanying paper) in the framework of a kinetic model. Taken together our data constitutes the first quantitative description of KcsA gating.

Accumulation of inactivation in a cloned transient K+ channel (AKv1.1a) of Aplysia

1995 ◽  
Vol 74 (3) ◽  
pp. 1248-1257 ◽  
Author(s):  
Y. Furukawa
Keyword(s):  
Short Interval ◽  
Low Frequency ◽  
K Channel ◽  
Amino Terminal ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
Inactivation Gating ◽  
A Cell ◽  
K Concentration ◽  
External K

1. Inactivation of a cloned Aplysia K+ channel, AKv1.1a, expressed in Xenopus oocytes was examined by a cell-attached macropatch recording. A fast macroscopic inactivation (the time constant for decay was in the range of 20-40 ms) in response to a depolarizing command pulse was insensitive to the concentration of external K+ (2-100 mM KCl). 2. By contrast, recovery from inactivation was extremely slow and dependent on external K+. When the concentration of external KCl was 2-3 mM, a patched membrane had to be held at hyperpolarized potential for > 40 s for a full recovery. The recovery was greatly accelerated if external K+ concentration was increased. A tail current following a command pulse long enough to inactivate most of the channels showed a marked rising phase. 3. A consequence of the slow recovery from inactivation was that AKv1.1a showed a marked accumulation of the inactivation following repetitive pulses, even at low frequency (< 0.1 Hz). When two depolarizing pulses were applied at a short interval, the current during a second pulse was smaller than the current at the end of the preceding pulse. This is a phenomenon called "cumulative inactivation." The onset and the extent of cumulative inactivation of AKv1.1a were voltage dependent but relatively insensitive to external K+ concentration. An amino terminal deletion mutant of AKv1.1a that lacks the fast N-type inactivation did not show cumulative inactivation. 4. These results suggest that the inactivation gating by the amino terminal region of AKv1.1a has a similarity to open-channel blockade, and that the cumulative inactivation can also be dependent on the amino terminal cytoplasmic domain of K+ channels.

Potassium loss from hypoxic myocardium: influence of external K concentration

1987 ◽  
Vol 65 (5) ◽  
pp. 861-866 ◽  
Author(s):  
Normand Leblanc ◽  
Elena Ruiz-Ceretti ◽  
Denis Chartier
Keyword(s):  
Electrical Activity ◽  
Sodium Content ◽  
Total Water ◽  
Tissue Samples ◽  
Normal Medium ◽  
High K ◽  
Voltage Dependent ◽  
External Potassium ◽  
K Concentration ◽  
External K

The influence of external potassium Ko and tetraethylammonium on the cellular K content of hypoxic myocardium was investigated. Perfused rabbit hearts were submitted to 60 min hypoxia in medium containing 5 mM K throughout or either low (1.5 mM) or high (10 mM) K during the last 20 min of hypoxia. Paced electrical activity (2.5 Hz) was kept throughout the experiments. Tissue samples excised from the left ventricle were analyzed for total water, inulin space, and Na and K content. Lowering Ko to 1.5 mM increased both K loss and Na accumulation. Addition of 3.5 mM RbCl under these conditions reversed Na accumulation to levels found for hypoxia in normal medium but did not modify the cellular K loss. Tetraethylammonium (10 mM) did not alter Na accumulation but partly prevented the decrease in K content produced by hypoxia. A similar effect was observed by increasing Ko to 10 mM. At this high Ko prolongation of hypoxia did not enhance K loss. Abolition of electrical activity by TTX in a high K solution prevented K loss and reduced the sodium content. These results are consistent with the view that voltage-dependent channels are implicated in the K loss induced by hypoxia or ischemia. Furthermore, they indicate that the K loss may be modulated by external K because of the influence of the electrochemical gradient on passive K efflux and thus provide an explanation for the existence of a plateau in the early extracellular K accumulation observed during cardiac ischemia.

Calcium channels and control of cytosolic calcium in rat and bovine zona glomerulosa cells

1992 ◽  
Vol 262 (3) ◽  
pp. C598-C606 ◽  
Author(s):  
S. J. Quinn ◽  
U. Brauneis ◽  
D. L. Tillotson ◽  
M. C. Cornwall ◽  
G. H. Williams
Keyword(s):  
Membrane Potential ◽  
Voltage Clamp ◽  
Cell Voltage ◽  
Cytosolic Calcium ◽  
Zona Glomerulosa ◽  
Voltage Dependent ◽  
Low Threshold ◽  
K Concentration ◽  
Ca2 Channels ◽  
External K

Rat and bovine adrenal zona glomerulosa (ZG) cells possess a low-threshold, voltage-dependent Ca2+ current that was characterized using whole cell voltage clamp techniques. Activation of this current is observed at membrane potentials above -80 mV with maximal peak Ca2+ current elicited near -30 mV. Inactivation of the Ca2+ current was half-maximal between -74 and -58 mV, depending on the external Ca2+ concentration and was nearly complete at -40 mV. The voltage dependency of the current indicates that a calcium current could be sustained at membrane potentials between -80 and -40 mV and thereby elevates cytosolic calcium (Cai) levels. Under basal conditions, Cai is stable in single rat ZG cells, whereas more than half of the bovine ZG cells produce repeated Cai transients. These Cai transients, which are blocked by removal of external Ca2+ or addition of Ni2+, are likely due to repetitive electrical activity in bovine ZG cells. Cai responses can be elicited by small increases in external K+ concentration (5-10 mM) in both rat and bovine ZG cells, indicating the opening of low-threshold Ca2+ channels. However, these Cai changes remain robust at high external K+ concentrations (20-40 mM). In experiments combining Cai measurements and whole cell voltage clamp, a steep dependence of Cai on membrane potential was revealed beginning at depolarizing voltages near a holding membrane potential of -80 mV. A maximal increase in Cai occurred near -30 mV (equivalent to an external K+ concentration of 40 mM), a membrane voltage at which sustained current through low-threshold Ca2+ channels should be negligible. These data raise the possibility of additional voltage-dependent pathways for Ca2+ influx.

scholarly journals Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

2008 ◽  
Vol 411 (3) ◽  
pp. 523-530 ◽  
Author(s):  
Gary S. Laco ◽  
Yves Pommier
Keyword(s):  
Amino Acids ◽  
Topoisomerase I ◽  
Electrostatic Interactions ◽  
Functional Domain ◽  
Site Mutation ◽  
Supercoiled Dna ◽  
Terminal Domain ◽  
Tryptophan Residues ◽  
N Terminus

Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp203/Trp205/Trp206) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan ‘anchor’ was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.

scholarly journals Factors affecting the appearance of the hump charge movement component in frog cut twitch fibers.

1991 ◽  
Vol 98 (2) ◽  
pp. 315-347 ◽  
Author(s):  
C S Hui
Keyword(s):  
Time Course ◽  
Extreme Case ◽  
Complete Recovery ◽  
Creatine Phosphate ◽  
Boltzmann Distribution ◽  
Distribution Functions ◽  
Charge Movement ◽  
Factors Affecting ◽  
Gap Technique ◽  
Voltage Dependent

Charge movement was measured in frog cut twitch fibers with the double Vaseline gap technique. Five manipulations listed below were applied to investigate their effects on the hump component (I gamma) in the ON segments of TEST minus CONTROL current traces. When external Cl-1 was replaced by MeSO3- to eliminate Cl current, I gamma peaked earlier due to a few millivolts shift of the voltage dependence of I gamma kinetics in the negative direction. The Q-V plots in the TEA.Cl and TEA.MeSO3 solutions were well fitted by a sum of two Boltzmann distribution functions. The more steeply voltage-dependent component (Q gamma) had a V approximately 6 mV more negative in the TEA.MeSO3 solution than in the TEA.Cl solution. These voltage shifts were partially reversible. When creatine phosphate in the end pool solution was removed, the I gamma hump disappeared slowly over the course of 20-30 min, partly due to a suppression of Q gamma. The hump reappeared when creatine phosphate was restored. When 0.2-1.0 mM Cd2+ was added to the center pool solution to block inward Ca current, the I gamma hump became less prominent due to a prolongation in the time course of I gamma but not to a suppression of Q gamma. When the holding potential was changed from -90 to -120 mV, the amplitude of I beta was increased, thereby obscuring the I gamma hump. Finally, when a cut fiber was stimulated repetitively, I gamma lost its hump appearance because its time course was prolonged. In an extreme case, a 5-min resting interval was insufficient for a complete recovery of the waveform. In general, a stimulation rate of once per minute had a negligible effect on the shape of I gamma. Of the five manipulations, MeSO3- has the least perturbation on the appearance of I gamma and is potentially a better substitute for Cl- than SO2-(4) in eliminating Cl current if the appearance of the I gamma hump is to be preserved.

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