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.

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 Mutations within the P-Loop of Kir6.2 Modulate the Intraburst Kinetics of the Atp-Sensitive Potassium Channel

2001 ◽  
Vol 118 (4) ◽  
pp. 341-353 ◽  
Author(s):  
Peter Proks ◽  
Charlotte E. Capener ◽  
Phillippa Jones ◽  
Frances M. Ashcroft
Keyword(s):  
Katp Channel ◽  
Single Channel ◽  
Katp Channels ◽  
Burst Duration ◽  
Open Probability ◽  
Closed Intervals ◽  
Open Time ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Kinetics Of

The ATP-sensitive potassium (KATP) channel exhibits spontaneous bursts of rapid openings, which are separated by long closed intervals. Previous studies have shown that mutations at the internal mouth of the pore-forming (Kir6.2) subunit of this channel affect the burst duration and the long interburst closings, but do not alter the fast intraburst kinetics. In this study, we have investigated the nature of the intraburst kinetics by using recombinant Kir6.2/SUR1 KATP channels heterologously expressed in Xenopus oocytes. Single-channel currents were studied in inside-out membrane patches. Mutations within the pore loop of Kir6.2 (V127T, G135F, and M137C) dramatically affected the mean open time (τo) and the short closed time (τC1) within a burst, and the number of openings per burst, but did not alter the burst duration, the interburst closed time, or the channel open probability. Thus, the V127T and M137C mutations produced longer τo, shorter τC1, and fewer openings per burst, whereas the G135F mutation had the opposite effect. All three mutations also reduced the single-channel conductance: from 70 pS for the wild-type channel to 62 pS (G135F), 50 pS (M137C), and 38 pS (V127T). These results are consistent with the idea that the KATP channel possesses a gate that governs the intraburst kinetics, which lies close to the selectivity filter. This gate appears to be able to operate independently of that which regulates the long interburst closings.

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 Inhibitors of asparagine-linked oligosaccharide processing alter the kinetics of the nicotinic acetylcholine receptor.

1989 ◽  
Vol 93 (5) ◽  
pp. 765-783 ◽  
Author(s):  
M Covarrubias ◽  
C Kopta ◽  
J H Steinbach
Keyword(s):  
Single Channel ◽  
Response Curves ◽  
Nicotinic Acetylcholine ◽  
Low Concentrations ◽  
Oligosaccharide Processing ◽  
Processing Step ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Kinetics Of ◽  
Ach Receptors

We used selective inhibitors of the asparagine-linked oligosaccharide processing pathway to study the effect of sugar trimming on the functional properties of the nicotinic acetylcholine (ACh) receptor expressed in clonal mammalian BC3H-1 cells. Inhibitors of initial steps of the processing pathway (1-deoxynojirimycin[DNJ] and castanospermine[CS]) reduced the density of ACh receptors on the cell surface (3- to 5-fold) but their responsiveness to ACh was more reduced (5- to 10-fold). These results suggest that the function of the ACh receptor was altered. When the ACh receptors were expressed in the presence of DNJ or CS, analysis of ACh-evoked single-channel currents (-100 mV and 11 degrees C) revealed an approximate threefold reduction in the opening rate (control: 600-650 s(-1)), treated: 130-250 s(-1)) and an approximate twofold reduction in the rate of agonist dissociation (control: 900-1,000 s(-1), treated: 400-500 s(-1)). In addition, the proportion of brief duration bursts (tau = 50-100 microseconds) was increased (1.5- to 2-fold) by treatments with DNJ or CS. In contrast, an inhibitor of a late processing step (swainsonine) did not produce such alterations. The single-channel conductance was not altered by any of the three inhibitors, and the slopes of log-log dose-response curves at low concentrations and desensitization did not appear to be affected. Each inhibitor altered the electrophoretic mobility of the ACh receptor subunits. We conclude that early sugar trimming can influence the kinetics of the nicotinic ACh receptor in BC3H-1 cells.

scholarly journals Mechanism of gating of T-type calcium channels.

1990 ◽  
Vol 96 (3) ◽  
pp. 603-630 ◽  
Author(s):  
C F Chen ◽  
P Hess
Keyword(s):  
Single Channel ◽  
Ca Channels ◽  
3T3 Fibroblasts ◽  
Channel Inactivation ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
Single Burst ◽  
Opening And Closing ◽  
Rate Limiting ◽  
Kinetics Of

We have analyzed the gating kinetics of T-type Ca channels in 3T3 fibroblasts. Our results show that channel closing, inactivation, and recovery from inactivation each include a voltage-independent step which becomes rate limiting at extreme potentials. The data require a cyclic model with a minimum of two closed, one open, and two inactivated states. Such a model can produce good fits to our data even if the transitions between closed states are the only voltage-dependent steps in the activating pathway leading from closed to inactivated states. Our analysis suggests that the channel inactivation step, as well as the direct opening and closing transitions, are not intrinsically voltage sensitive. Single-channel recordings are consistent with this scheme. As expected, each channel produces a single burst per opening and then inactivates. Comparison of the kinetics of T-type Ca current in fibroblasts and neuronal cells reveals significant differences which suggest that different subtypes of T-type Ca channels are expressed differentially in a tissue specific manner.

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.

Sign in / Sign up

Export Citation Format

Share Document