scholarly journals A Quantitative Description of KcsA Gating II: Single-Channel Currents

2007 ◽  
Vol 130 (5) ◽  
pp. 479-496 ◽  
Author(s):  
Sudha Chakrapani ◽  
Julio F Cordero-Morales ◽  
Eduardo Perozo
Keyword(s):  
Single Channel ◽  
Quantitative Description ◽  
Preceding Paper ◽  
Burst Frequency ◽  
Voltage Dependent ◽  
Subconductance States ◽  
Kinetic Transitions ◽  
Single Channel Currents ◽  
Open States ◽  
Channel Currents

The kinetic transitions of proton-activated WT KcsA and the noninactivating E71A mutant were studied at the single-channel level in purified, liposome-reconstituted preparations. Single-channel currents were recorded using patch-clamp techniques under nonstationary and steady-state conditions. Maximum-likelihood analyses reveal that the key influence of acidic pH is to increase the frequency of bursting without an effect on the intraburst open and closed dwell times, consistent with the finding from macroscopic currents that protons promote activation without a significant effect on inactivation. However, in steady-conditions of pH, voltage not only alters the burst frequency but also affects their properties, such as the frequency of the flickers and the dwell times of the closed and open states. This is to be expected if voltage modulates pathways connecting open and inactivated states. Upon opening, KcsA can enter at least two closed states that are not part of the activation pathway. The frequency and duration of these closed states was found to be voltage dependent and therefore these are likely to represent short-lived inactivated states. Single-channel recordings of WT KcsA also show varying propensity for the presence of subconductance states. The probability of occurrence of these states did not show clear modulation by voltage or pH and their origin remains unclear and a focus for further investigation. A kinetic model is proposed to describe the gating events in KcsA that recapitulates its macroscopic and single-channel behavior. The model has been constrained by the single-channel analyses presented in this work along with data from macroscopic currents in the preceding paper.

scholarly journals IKs ion-channel pore conductance can result from individual voltage sensor movements

2019 ◽  
Vol 116 (16) ◽  
pp. 7879-7888 ◽  
Author(s):  
Maartje Westhoff ◽  
Jodene Eldstrom ◽  
Christopher I. Murray ◽  
Emely Thompson ◽  
David Fedida
Keyword(s):  
Single Channel ◽  
Voltage Sensor ◽  
Open Probability ◽  
Voltage Sensors ◽  
Repolarization Reserve ◽  
Accessory Subunits ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Open States ◽  
Channel Currents

The IKs current has an established role in cardiac action potential repolarization, and provides a repolarization reserve at times of stress. The underlying channels are formed from tetramers of KCNQ1 along with one to four KCNE1 accessory subunits, but how these components together gate the IKs complex to open the pore is controversial. Currently, either a concerted movement involving all four subunits of the tetramer or allosteric regulation of open probability through voltage-dependent subunit activation is thought to precede opening. Here, by using the E160R mutation in KCNQ1 or the F57W mutation in KCNE1 to prevent or impede, respectively, voltage sensors from moving into activated conformations, we demonstrate that a concerted transition of all four subunits after voltage sensor activation is not required for the opening of IKs channels. Tracking voltage sensor movement, via [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) modification and fluorescence recordings, shows that E160R-containing voltage sensors do not translocate upon depolarization. E160R, when expressed in all four KCNQ1 subunits, is nonconducting, but if one, two, or three voltage sensors contain the E160R mutation, whole-cell and single-channel currents are still observed in both the presence and absence of KCNE1, and average conductance is reduced proportional to the number of E160R voltage sensors. The data suggest that KCNQ1 + KCNE1 channels gate like KCNQ1 alone. A model of independent voltage sensors directly coupled to open states can simulate experimental changes in IKs current kinetics, including the nonlinear depolarization of the conductance–voltage (G–V) relationship, and tail current acceleration as the number of nonactivatable E160R subunits is increased.

Potassium channels activated by GABAB agonists and serotonin in cultured hippocampal neurons

1994 ◽  
Vol 71 (6) ◽  
pp. 2570-2575 ◽  
Author(s):  
L. S. Premkumar ◽  
P. W. Gage
Keyword(s):  
Potassium Channels ◽  
Hippocampal Neurons ◽  
Single Channel ◽  
Gamma Aminobutyric Acid ◽  
Kinetic Behavior ◽  
Open Time ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Cultured Hippocampal Neurons

1. Single-channel currents were recorded in cell-attached patches on cultured hippocampal neurons in response to gamma-aminobutyric acid-B (GABAB) agonists or serotonin applied to the cell surface outside the patch area. 2. The channels activated by GABAB agonists and serotonin were potassium selective but had a different conductance and kinetic behavior. Channels activated by GABAB agonists had a higher conductance, longer open-time, and longer burst-length than channels activated by serotonin. 3. The kinetic behavior of channels activated by GABAB agonists varied with potential whereas channels activated by serotonin did not show voltage-dependent changes in kinetics. 4. In a few cell-attached patches, both types of channel were activated when the cell was exposed to GABA together with serotonin. 5. It was concluded that GABAB agonists and serotonin activate different potassium channels in the soma of cultured hippocampal neurons.

Rectification of muscarinic K+ current by magnesium ion in guinea pig atrial cells

1987 ◽  
Vol 253 (1) ◽  
pp. H210-H214
Author(s):  
M. Horie ◽  
H. Irisawa
Keyword(s):  
Guinea Pig ◽  
Action Potentials ◽  
Single Channel ◽  
Outward Current ◽  
Atrial Cells ◽  
Voltage Dependent ◽  
K Current ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Low K

Rectifying properties of the acetylcholine (ACh)-sensitive K+ channels were studied using a patch-clamp method in single atrial cells prepared enzymatically from adult guinea pig hearts. In the presence of micromolar concentration of nonhydrolyzable guanosine 5'-triphosphate (GTP) analogue 5'-guanylylimidodiphosphate (GppNHp) and the absence of Mg2+ at the inner surface of patch membrane [( Mg2+]i), the channel activity recovered in inside-out patch condition. The single channel conductance became ohmic between -80 and +80 mV (symmetrical 150 mM K+ solutions). The rapid relaxation of outward single channel currents was disclosed on a depolarization. [Mg2+]i blocked the outward current through the channel dose- and voltage-dependently and also induced a dose-dependent increase in the channel activation. The apparent paradoxical role of [Mg2+]i is important for the cholinergic control in the heart; voltage-dependent Mg block ensures a low K+ conductance of cell membrane at the plateau of action potentials during the exposure to ACh, thereby slowing the heart rate without unfavorable shortening of the action potentials.

scholarly journals Divalent Cation Interactions with Light-Dependent K Channels

1999 ◽  
Vol 114 (5) ◽  
pp. 653-672 ◽  
Author(s):  
Enrico Nasi ◽  
Maria del Pilar Gomez
Keyword(s):  
Binding Site ◽  
Single Channel ◽  
Divalent Cations ◽  
Light Response ◽  
Relaxation Methods ◽  
Physiological Conditions ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Single Binding Site

The light-dependent K conductance of hyperpolarizing Pecten photoreceptors exhibits a pronounced outward rectification that is eliminated by removal of extracellular divalent cations. The voltage-dependent block by Ca2+ and Mg2+ that underlies such nonlinearity was investigated. Both divalents reduce the photocurrent amplitude, the potency being significantly higher for Ca2+ than Mg2+ (K1/2 ≈ 16 and 61 mM, respectively, at Vm = −30 mV). Neither cation is measurably permeant. Manipulating the concentration of permeant K ions affects the blockade, suggesting that the mechanism entails occlusion of the permeation pathway. The voltage dependency of Ca2+ block is consistent with a single binding site located at an electrical distance of δ ≈ 0.6 from the outside. Resolution of light-dependent single-channel currents under physiological conditions indicates that blockade must be slow, which prompted the use of perturbation/relaxation methods to analyze its kinetics. Voltage steps during illumination produce a distinct relaxation in the photocurrent (τ = 5–20 ms) that disappears on removal of Ca2+ and Mg2+ and thus reflects enhancement or relief of blockade, depending on the polarity of the stimulus. The equilibration kinetics are significantly faster with Ca2+ than with Mg2+, suggesting that the process is dominated by the “on” rate, perhaps because of a step requiring dehydration of the blocking ion to access the binding site. Complementary strategies were adopted to investigate the interaction between blockade and channel gating: the photocurrent decay accelerates with hyperpolarization, but the effect requires extracellular divalents. Moreover, conditioning voltage steps terminated immediately before light stimulation failed to affect the photocurrent. These observations suggest that equilibration of block at different voltages requires an open pore. Inducing channels to close during a conditioning hyperpolarization resulted in a slight delay in the rising phase of a subsequent light response; this effect can be interpreted as closure of the channel with a divalent ion trapped inside.

scholarly journals Purified and unpurified sodium channels from eel electroplax in planar lipid bilayers.

1987 ◽  
Vol 90 (3) ◽  
pp. 375-395 ◽  
Author(s):  
E Recio-Pinto ◽  
D S Duch ◽  
S R Levinson ◽  
B W Urban
Keyword(s):  
Sodium Channel ◽  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Planar Bilayers ◽  
Electric Eel ◽  
Voltage Dependent ◽  
Single Channel Activity ◽  
Single Channel Currents ◽  
Channel Currents

Highly purified sodium channel protein from the electric eel, Electrophorus electricus, was reconstituted into liposomes and incorporated into planar bilayers made from neutral phospholipids dissolved in decane. The purest sodium channel preparations consisted of only the large, 260-kD tetrodotoxin (TTX)-binding polypeptide. For all preparations, batrachotoxin (BTX) induced long-lived single-channel currents (25 pS at 500 mM NaCl) that showed voltage-dependent activation and were blocked by TTX. This block was also voltage dependent, with negative potentials increasing block. The permeability ratios were 4.7 for Na+:K+ and 1.6 for Na+:Li+. The midpoint for steady state activation occurred around -70 mV and did not shift significantly when the NaCl concentration was increased from 50 to 1,000 mM. Veratridine-induced single-channel currents were about half the size of those activated by BTX. Unpurified, nonsolubilized sodium channels from E. electricus membrane fragments were also incorporated into planar bilayers. There were no detectable differences in the characteristics of unpurified and purified sodium channels, although membrane stability was considerably higher when purified material was used. Thus, in the eel, the large, 260-kD polypeptide alone is sufficient to demonstrate single-channel activity like that observed for mammalian sodium channel preparations in which smaller subunits have been found.

scholarly journals Guanidine block of single channel currents activated by acetylcholine.

1986 ◽  
Vol 88 (5) ◽  
pp. 635-650 ◽  
Author(s):  
T M Dwyer
Keyword(s):  
Driving Force ◽  
Single Channel ◽  
Ion Flow ◽  
Channel Current ◽  
Outward Currents ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Reversal Potentials ◽  
Patch Clamp Method

The acetylcholine-activated channel of chick myotube was studied using the patch-clamp method. Single channel current amplitudes were measured between -300 and +250 mV in solutions containing the permeant ions Cs+ and guanidine (G+). G+ has a relative permeability, PG/PCs, of 1.6, but carries no more than half the current that Cs+ does, with an equivalent electrochemical driving force. Experiments using G+ revealed an asymmetry of the acetylcholine-activated channel, with G+ being more effective at reducing Cs+ currents when added to the outside than when added to the inside. The block caused by outside, but not inside, G+ was evident for both inward and outward currents. The block caused by outside G+ was voltage dependent, first increasing and then being partially relieved when the driving force was made more negative. Experiments with mixtures of Cs+ and G+ revealed anomalously low magnitudes for reversal potentials, relative to predictions based on the Goldman-Hodgkin-Katz equation. These findings are consistent with a two-well, three-barrier Eyring rate model for ion flow, and demonstrate that a highly permeant ion, guanidine, can block asymmetrically by acting from within the voltage field of the acetylcholine-activated channel.

Single calcium channel currents of arterial smooth muscle at physiological calcium concentrations

1992 ◽  
Vol 263 (5) ◽  
pp. C948-C952 ◽  
Author(s):  
M. Gollasch ◽  
J. Hescheler ◽  
J. M. Quayle ◽  
J. B. Patlak ◽  
M. T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Single Channel ◽  
Membrane Potentials ◽  
Ca Channel ◽  
Ca Channels ◽  
Arterial Smooth Muscle ◽  
Voltage Dependent ◽  
High Concentrations ◽  
Single Channel Currents ◽  
Channel Currents

Entry of Ca through voltage-dependent Ca channels is an important regulator of the function of smooth muscle, cardiac muscle, and neurons. Although Ca channels have been extensively studied since the first descriptions of Ca action potentials (P. Fatt and B. Katz. J. Physiol. Lond. 120: 171-204, 1953), the permeation rate of Ca through single Ca channels has not been measured directly under physiological conditions. Instead, single Ca channels have typically been examined using high concentrations (80-110 mM) of another divalent charge carrier, Ba, so as to maximize the amplitude of the single-channel currents. Calculations of unitary currents at 2 mM Ca indicated that the single-channel currents would be immeasurably small (i.e., < 0.1 pA). We provide here the first direct measurements of single Ca channel currents at a physiological Ca concentration. Contrary to earlier estimates, we have found that currents through single Ca channels in arterial smooth muscle are 0.1-0.3 pA at 2 mM Ca and physiological membrane potentials. These relatively large unitary currents permit direct measurement of Ca channel properties under conditions that do not distort their function. Our data also indicate that Ca permeates these channels at relatively high rates in physiological Ca concentrations and membrane potentials.

scholarly journals Multiple effects of KPQ deletion mutation on gating of human cardiac Na+ channels expressed in mammalian cells

1998 ◽  
Vol 274 (5) ◽  
pp. H1643-H1654 ◽  
Author(s):  
Rashmi Chandra ◽  
C. Frank Starmer ◽  
Augustus O. Grant
Keyword(s):  
Mammalian Cells ◽  
Single Channel ◽  
Deletion Mutation ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
Rate Of Recovery ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Kinetics Of ◽  
Rate Of Activation

Several aspects of the effect of the KPQ deletion mutation on Na+ channel gating remain unresolved. We have analyzed the kinetics of the early and late currents by recording whole cell and single-channel currents in a human embryonic kidney (HEK) cell line (HEK293) expressing wild-type and KPQ deletion mutation in cardiac Na+ channels. The rate of inactivation increased three- to fivefold between −40 and −80 mV in the mutant channel. The rate of recovery from inactivation was increased twofold. Two modes of gating accounted for the late current: 1) isolated brief openings with open times that were weakly voltage dependent and the same as the initial transient and 2) bursts of opening with highly voltage-dependent prolonged open times. Latency to first opening was accelerated, suggesting an acceleration of the rate of activation. The ΔKPQ mutation has multiple effects on activation and inactivation. The aggregate effects may account for the increased susceptibility to arrhythmias.

scholarly journals Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle.

1984 ◽  
Vol 84 (1) ◽  
pp. 1-23 ◽  
Author(s):  
A L Blatz ◽  
K L Magleby
Keyword(s):  
Dissociation Constant ◽  
Single Channel ◽  
Reversal Potential ◽  
Ion Concentration ◽  
Dependent Manner ◽  
Apparent Dissociation Constant ◽  
Rat Muscle ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Channel Currents

The conductance and selectivity of the Ca-activated K channel in cultured rat muscle was studied. Shifts in the reversal potential of single channel currents when various cations were substituted for Ki+ were used with the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The selectivity was Tl+ greater than K+ greater than Rb+ greater than NH4+, with permeability ratios of 1.2, 1.0, 0.67, and 0.11. Na+, Li+, and Cs+ were not measurably permeant, with permeabilities less than 0.05 that of K+. Currents with the various ions were typically less than expected on the basis of the permeability ratios, which suggests that the movement of an ion through the channel was not independent of the other ions present. For a fixed activity of Ko+ (77 mM), plots of single channel conductance vs. activity of Ki+ were described by a two-barrier model with a single saturable site. This observation, plus the finding that the permeability ratios of Rb+ and NH+4 to K+ did not change with ion concentration, is consistent with a channel that can contain a maximum of one ion at any time. The empirically determined dissociation constant for the single saturable site was 100 mM, and the maximum calculated conductance for symmetrical solutions of K+ was 640 pS. TEAi+ (tetraethylammonium ion) reduced single channel current amplitude in a voltage-dependent manner. This effect was accounted for by assuming voltage-dependent block by TEA+ (apparent dissociation constant of 60 mM at 0 mV) at a site located 26% of the distance across the membrane potential, starting at the inner side. TEAo+ was much more effective in reducing single channel currents, with an apparent dissociation constant of approximately 0.3 mM.

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