scholarly journals Reconstitution of the ATP-sensitive potassium channel of skeletal muscle. Activation by a G protein-dependent process.

1989 ◽  
Vol 94 (3) ◽  
pp. 445-463 ◽  
Author(s):  
L Parent ◽  
R Coronado
Keyword(s):  
Skeletal Muscle ◽  
G Protein ◽  
Muscle Activation ◽  
Open Channel ◽  
Single Channel ◽  
Rabbit Skeletal Muscle ◽  
S 100 ◽  
Internal Side ◽  
Gamma S ◽  
Channel Properties

Potassium channels inhibited by adenosine-5'-trisphosphate, K(ATP), found in the transverse tubular membrane of rabbit skeletal muscle were studied using the planar bilayer recording technique. In addition to the single-channel properties of K(ATP) we report its regulation of Mg2+ and by the guanosine-5'-trisphosphate analogue, GTP-y(gamma)-S. The K(ATP) channel (a) has a conductance of 67 pS in 250 mM internal, 50 mM external KCl, and rectifies weakly at holding potentials more positive than 50 mV, (b) is not activated by internal Ca2+ or membrane depolarization, (c) has a permeability ratio PK/PNa greater than 50, and (d) is inhibited by millimolar internal ATP. Activity of K(ATP), measured as open channel probability as a function of time, was unstable at all holding potentials and decreases continuously within a few minutes after a recording is initiated. After a decrease in activity, GTP-y-S (100 microM) added to the internal side reactivated K(ATP) channels but only transiently. In the presence of internal 1 mM Mg2+, GTP-y-S produced a sustained reactivation lasting 20-45 min. Incubation of purified t-tubule vesicles with AlF4 increased the activity of K(ATP) channels, mimicking the effect of GTP-y-S. The effect of AlF4 and the requirement of GTP-y-S plus Mg2+ for sustained channel activation suggests that a nucleotide-binding G protein regulates ATP-sensitive K channels in the t-tuble membrane of rabbit skeletal muscle.

The neuropeptide FMRFa both inhibits and enhances the Ca2+ current in dissociated Helix neurons via independent mechanisms

1991 ◽  
Vol 65 (6) ◽  
pp. 1517-1527 ◽  
Author(s):  
J. L. Yakel
Keyword(s):  
G Protein ◽  
Cell Voltage ◽  
Inhibitory Response ◽  
Central Neurons ◽  
S 100 ◽  
Helix Neurons ◽  
20 Nm ◽  
Gamma S ◽  
Ca2 Channels ◽  
Rate Of Activation

1. The modulation of the voltage-activated Ca2+ current by the neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFa) was investigated in dissociated central neurons from Helix aspersa using whole-cell voltage-clamp recording techniques. External Ba2+ was always used as the charge carrier in this study, and the intracellular Ca2+ concentration was buffered to 20 nM with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). 2. Run-down of the Ca2+ currents was not a problem as long as the neurons were dialyzed with a patch electrode filling solution containing ATP (1 or 2 mM). In ATP-dialyzed neurons, the rate of inactivation of the calcium current increased with time without any significant change in the rate of activation. However, when neurons were dialyzed with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 100 microM; with ATP), the rate of inactivation decreased with time. There was no effect of GTP gamma S on the rate of activation of the Ca2+ current. This suggests that guanosine 5'-triphosphate (GTP)-binding proteins (G proteins) are able to modulate the rate of inactivation of the Ca2+ current in Helix neurons. 3. FMRFa both decreased and enhanced the amplitude of the Ca2+ current in these neurons. This inhibition was observed in most neurons, while the enhancement was observed in 20% of the neurons. Although the enhancement usually was preceded by the inhibitory response, sometimes the enhancement was observed separately. 4. The FMRFa-induced inhibition of the Ca2+ current usually consisted of a decrease in both the amplitude and the rate of inactivation of this current, effects that were reduced as the membrane potential was stepped to more depolarized potentials. A pertussis toxin (PTX)-sensitive G protein mediated this response, whereas no evidence was found to suggest the involvement of any known intracellular messenger. Therefore this inhibition may have resulted from a direct coupling between the FMRFa receptor and the Ca2+ channels via a PTX-sensitive G protein. 5. Arachidonic acid (100 microM) irreversibly reduced the amplitude of the Ca2+ current, but it did not alter the relative inhibition of this current by FMRFa. 6. The FMRFa-induced enhancement of the Ca2+ current was difficult to study because it was observed infrequently, and was rarely observed independently of the FMRFa-induced inhibitory response. In addition, the ability of FMRFa to enhance this current usually disappeared with time.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Modeling Macroscopic Currents of Ion Channels

2021 ◽  
Author(s):  
Di Wu
Keyword(s):  
Open Channel ◽  
Single Channel ◽  
Ion Permeation ◽  
Model Studies ◽  
Proposed Model ◽  
Current Voltage ◽  
Improved Model ◽  
Boltzmann Model ◽  
Channel Properties ◽  
Current Voltage Relation

Ion-channel functions are often studied by the current-voltage relation, which is commonly fitted by the Boltzmann equation, a powerful model widely used nowadays. However, the Boltzmann model is restricted to a two-state ion-permeation process. Here we present an improved model that comprises a flexible number of states and incorporates both the single-channel conductance and the open-channel probability. Employing the channel properties derived from the single-channel recording experiments, the proposed model is able to describe various current-voltage relations, especially the reversal ion-permeation curves showing the inward- and outward-rectifications. We demonstrate the applicability of the proposed model using the published patch-clamp data of BK and MthK potassium channels, and discuss the similarity of the two channels based on the model studies.

scholarly journals Single-channel properties of skeletal muscle ryanodine receptor pore Δ4923FF4924 in two brothers with a lethal form of fetal akinesia

Cell Calcium ◽  
2020 ◽  
Vol 87 ◽  
pp. 102182 ◽  
Author(s):  
Le Xu ◽  
Frederike L. Harms ◽  
Venkat R. Chirasani ◽  
Daniel A. Pasek ◽  
Fanny Kortüm ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Ryanodine Receptor ◽  
Single Channel ◽  
Channel Properties

scholarly journals Single-channel properties of the recombinant skeletal muscle Ca2+ release channel (ryanodine receptor)

1997 ◽  
Vol 73 (4) ◽  
pp. 1904-1912 ◽  
Author(s):  
S.R. Chen ◽  
P. Leong ◽  
J.P. Imredy ◽  
C. Bartlett ◽  
L. Zhang ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Ryanodine Receptor ◽  
Single Channel ◽  
Release Channel ◽  
Channel Properties

scholarly journals GTP-dependent regulation of myometrial KCa channels incorporated into lipid bilayers.

1990 ◽  
Vol 96 (2) ◽  
pp. 373-394 ◽  
Author(s):  
L Toro ◽  
J Ramos-Franco ◽  
E Stefani
Keyword(s):  
G Protein ◽  
Lipid Bilayers ◽  
Adrenergic Receptors ◽  
Single Channel ◽  
Channel Activity ◽  
Beta Adrenergic Receptors ◽  
Open Probability ◽  
Beta Adrenergic ◽  
Kca Channels ◽  
Gamma S

The regulation of calcium-activated K (KCa) channels by a G protein-mediated mechanism was studied. KCa channels were reconstituted in planar lipid bilayers by fusion of membrane vesicles from rat or pig myometrium. The regulatory process was studied by exploring the actions of GTP and GTP gamma S on single channel activity. KCa channels had a conductance of 260 +/- 6 pS (n = 25, +/- SE, 250/50 mM KCl gradient) and were voltage dependent. The open probability (Po) vs. voltage relationships were well fit by a Boltzmann distribution. The slope factor (11 mV) was insensitive to internal Ca2+. The half activation potential (V1/2) was shifted -70 mV by raising internal Ca2+ from pCa 6.2 to pCa 4. Addition of GTP or GTP gamma S activated channel activity only in the presence of Mg2+, a characteristic typical of G protein-mediated mechanisms. The Po increased from 0.18 +/- 0.08 to 0.49 +/- 0.07 (n = 7, 0 mV, pCa 6 to 6.8). The channel was also activated (Po increased from 0.03 to 0.37) in the presence of AMP-PNP, a nonphosphorylating ATP analogue, suggesting a direct G protein gating of KCa channels. Upon nucleotide activation, mean open time increased by a factor of 2.7 +/- 0.7 and mean closed time decreased by 0.2 +/- 0.07 of their initial values (n = 6). Norepinephrine (NE) or isoproterenol potentiated the GTP-mediated activation of KCa channels (Po increased from 0.17 +/- 0.06 to 0.35 +/- 0.07, n = 10). These results suggest that myometrium possesses beta-adrenergic receptors coupled to a GTP-dependent protein that can directly gate KCa channels. Furthermore, KCa channels, beta-adrenergic receptors, and G proteins can be reconstituted in lipid bilayers as a stable, functionally coupled, molecular complex.

scholarly journals Ionic interactions of Ba2+ blockades in the MthK K+ channel

2014 ◽  
Vol 144 (2) ◽  
pp. 193-200 ◽  
Author(s):  
Rui Guo ◽  
Weizhong Zeng ◽  
Hengjun Cui ◽  
Liping Chen ◽  
Sheng Ye
Keyword(s):  
Single Channel ◽  
K Channel ◽  
K Channels ◽  
X Ray ◽  
X Ray Crystallography ◽  
Functional Studies ◽  
Single File ◽  
Conduction Pathway ◽  
Internal Side ◽  
Channel Properties

The movement and interaction of multiple ions passing through in single file underlie various fundamental K+ channel properties, from the effective conduction of K+ ions to channel blockade by Ba2+ ions. In this study, we used single-channel electrophysiology and x-ray crystallography to probe the interactions of Ba2+ with permeant ions within the ion conduction pathway of the MthK K+ channel. We found that, as typical of K+ channels, the MthK channel was blocked by Ba2+ at the internal side, and the Ba2+-blocking effect was enhanced by external K+. We also obtained crystal structures of the MthK K+ channel pore in both Ba2+–Na+ and Ba2+–K+ environments. In the Ba2+–Na+ environment, we found that a single Ba2+ ion remained bound in the selectivity filter, preferably at site 2, whereas in the Ba2+–K+ environment, Ba2+ ions were predominantly distributed between sites 3 and 4. These ionic configurations are remarkably consistent with the functional studies and identify a molecular basis for Ba2+ blockade of K+ channels.

scholarly journals Subconductance block of single mechanosensitive ion channels in skeletal muscle fibers by aminoglycoside antibiotics.

1996 ◽  
Vol 107 (3) ◽  
pp. 433-443 ◽  
Author(s):  
B D Winegar ◽  
C M Haws ◽  
J B Lansman
Keyword(s):  
Skeletal Muscle ◽  
Open Channel ◽  
Single Channel ◽  
Muscle Fibers ◽  
Bimolecular Reaction ◽  
Aminoglycoside Antibiotics ◽  
Ion Flow ◽  
Channel Current ◽  
Skeletal Muscle Fibers ◽  
Drug Concentrations

The activity of single mechanosensitive channels was recorded from cell-attached patches on acutely isolated skeletal muscle fibers from the mouse. The experiments were designed to investigate the mechanism of channel block produced by externally applied aminoglycoside antibiotics. Neomycin and other aminoglycosides reduced the amplitude of the single-channel current at negative membrane potentials. The block was concentration-dependent, with a half-maximal concentration of approximately 200 microM. At high drug concentrations, however, block was incomplete with roughly one third of the current remaining unblocked. Neomycin also caused the channel to fluctuate between the open state and a subconductance level that was also roughly one third the amplitude of the fully open level. An analysis of the kinetics of the subconductance fluctuations was consistent with a bimolecular reaction between an aminoglycoside molecule and the open channel (kon = approximately 1 x 10(6) M-1s-1 and koff = approximately 400 s-1 at -60 mV). Increasing the external pH reduced both the rapid block of the open channel and the frequency of the subconductance fluctuations, as if both blocking actions were produced by a single active drug species with a pKa = approximately 7.5. The results are interpreted in terms of a mechanism in which an aminoglycoside molecule partially occludes ion flow through the channel pore.

scholarly journals Ion permeation through 5-hydroxytryptamine-gated channels in neuroblastoma N18 cells.

1990 ◽  
Vol 96 (6) ◽  
pp. 1177-1198 ◽  
Author(s):  
J Yang
Keyword(s):  
Single Channel ◽  
Cell Voltage ◽  
Ionic Currents ◽  
Organic Cations ◽  
Fluctuation Analysis ◽  
Divalent Ions ◽  
Apparent Permeability ◽  
S 100 ◽  
Specific Antagonist ◽  
Gamma S

Ionic currents induced by 5-hydroxytryptamine (5-HT) in cultured neuroblastoma N18 cells were studied using whole-cell voltage clamp. The response was blocked by 1-10 nM 5-HT3 receptor-specific antagonists MDL 7222 or ICS 205-930, but not by 1 microM 5-HT1/5-HT2 receptor antagonist spiperone or 5-HT2 receptor-specific antagonist ketanserin. These 5-HT3 receptors seem to be ligand-gated channels because the response (a) did not require internal ATP or GTP, (b) persisted with long internal dialysis of CsF (90 mM), A1F4- (100 microM), or GTP gamma S (100 microM), and (c) with ionophoretic delivery of 5-HT developed with a delay of less than 10 ms and rose to a peak in 34-130 ms. Fluctuation analysis yielded an apparent single-channel conductance of 593 fS. The relative permeabilities of the channel for a variety of ions were determined from reversal potentials. The channel was only weakly selective among small cations, with permeability ratios PX/PNa of 1.22, 1.10, 1.01, 1.00, and 0.99 for Cs+, K+, Li+, Na+, and Rb+, and 1.12, 0.79, and 0.73 for Ca2+, Ba2+, and Mg2+ (when studied in mixtures of 20 mM divalent ions and 120 mM N-methyl-D-glucamine). Apparent permeability ratios for the divalent ions decreased as the concentration of divalent ions was increased. Small monovalent organic cations were highly permeant. Large organic cations such as Tris and glucosamine were measurably permeant with permeability ratios of 0.20 and 0.08, and N-methyl-D-glucamine was almost impermeant. Small anions, NO3-, Cl-, and F-, were slightly permeant with permeability ratios of 0.08, 0.04, and 0.03. The results indicate that the open 5-HT3 receptor channel has an effective minimum circular pore size of 7.6 A and that ionic interactions in the channel may involve negative charges near the pore mouth.

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