scholarly journals Kinetic properties of single sodium channels in rat heart and rat brain.

1989 ◽  
Vol 93 (1) ◽  
pp. 85-99 ◽  
Author(s):  
G E Kirsch ◽  
A M Brown
Keyword(s):  
Cortical Neurons ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Na Channel ◽  
Kinetic Properties ◽  
Open Time ◽  
Excised Patches ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Near Threshold

Single Na channel currents were compared in ventricular myocytes and cortical neurons of neonatal rats using the gigaseal patch-clamp method to determine whether tissue-specific differences in gating can be detected at the single-channel level. Single-channel currents were recorded in cell-attached and excised membrane patches at test potentials of -70 to -20 mV and at 9-11 degrees C. In both cell-attached and excised patches brain Na channel mean open time progressively increased from less than 1 ms at -70 mV to approximately 2 ms at -20 mV. Near threshold, single openings with dispersed latencies were observed. By contrast, in cell-attached patches, heart Na channel mean open time peaked near -50 mV, was three times brain Na channel mean open time, and declined continuously to approximately 2 ms at -20 mV. Near threshold, openings occurred frequently usually as brief bursts lasting several milliseconds and rarely as prolonged bursts lasting tens of milliseconds. Unlike what occurs in brain tissue where excision did not change gating, in excised heart patches both the frequency of prolonged bursting and the mean open time of single units increased markedly. Brain and cardiac Na channels can therefore be distinguished on the basis of their mean open times and bursting characteristics.

scholarly journals Modification of Na channel gating by an alpha scorpion toxin from Tityus serrulatus.

1989 ◽  
Vol 93 (1) ◽  
pp. 67-83 ◽  
Author(s):  
G E Kirsch ◽  
A Skattebøl ◽  
L D Possani ◽  
A M Brown
Keyword(s):  
Cortical Neurons ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Neuroblastoma Cells ◽  
Na Channel ◽  
External Application ◽  
Whole Cell ◽  
Peak Current ◽  
Open Time ◽  
Tityus Serrulatus

The effects of TsIV-5, a toxin isolated from the Brazilian scorpion Tityus serrulatus, on whole-cell and single-channel Na currents were determined in N18 neuroblastoma cells. In whole-cell records at a test potential of -10 mV, external application of 500 nM TsIV-5 slowed inactivation 20-fold and increased peak current by about one-third without changing time-to-peak. Both the steady-state activation and inactivation curves were shifted to more negative potentials. Other alpha scorpion toxins produce similar effects but the single-channel mechanism is not known. TsIV-5 caused a voltage-dependent prolongation of mean single-channel open time such that at a test potential of -60 mV no change was observed, whereas at -20 mV mean open time increased about threefold and prolonged bursting was observed. Macroscopic current reconstructed from summed single-channel records showed a characteristic toxin-induced potentiation of peak current and a 20-fold slowing of the decay phase. TsIV-5 does not discriminate between tissue-specific Na channel subtypes. Prolonged open times and bursting were also observed in toxin-treated Na channels from rat ventricular myocytes, rat cortical neurons, and mouse skeletal muscle. The toxin effects are shown to be consistent with a kinetic model in which TsIV-5 selectively interferes with the ability of the channel to reach the inactivated state.

Oxantel-activated single channel currents in the muscle membrane of Ascaris suum

Parasitology ◽  
1995 ◽  
Vol 110 (4) ◽  
pp. 437-448 ◽  
Author(s):  
V. M. E. Dale ◽  
R. J. Martin
Keyword(s):  
Single Channel ◽  
Ascaris Suum ◽  
Muscle Membrane ◽  
Kinetic Properties ◽  
Voltage Sensitivity ◽  
Channel Block ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Single Channel Currents ◽  
Channel Currents

SUMMARYThe patch clamp technique was used to investigate the action of the anthelmintic drug, oxantel, on nicotinic acetylcholine receptor (nAChR) currents recorded from vesicles of the somatic muscle cells of the nematode parasite Ascaris suum. The amplitudes of the currents were analysed at different membrane potentials to determine the single channel conductance. Also the open and closed durations were measured to determine the kinetic properties of the activated channel. Oxantel activated single nAChR currents throughout a concentration range 10–100 μM, these currents were not observed with oxantel-free pipette solutions. The mean open time of the activated channels at a membrane potential of –75 mV and a concentration of 10 μM was 1·34 ms. At higher concentrations the open times were shorter and voltage sensitive, decreasing in duration on hyperpolarization, thus suggesting open channel block. The kinetics were analysed using a simple channel block model. The forward block rate, K + B, increased with increasing oxantel concentration but showed little increase as the membrane was hyperpolarized. K + B was 2·41×107 M−1s−1 – 50 mV and 2·64 × 107 M−1s−1 at – 100mV. The unblocking rate constant, K – B, did exhibit voltage sensitivity being 443·6 s−1 at – 50 mV and 86·8 s−1 at –100 mV. Thus the blocking dissociation constant KB (= K – B/K + B) was 18·5 μM at –50 mV and 3·3 μM at –100 mV. The simple channel block scheme was found to be insufficient to explain fully the observations made; reasons for this are discussed.

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.

scholarly journals Stretch-activated whole cell currents in adult rat cardiac myocytes

2000 ◽  
Vol 278 (2) ◽  
pp. H548-H557 ◽  
Author(s):  
Tao Zeng ◽  
Glenna C. L. Bett ◽  
Frederick Sachs
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Reversal Potential ◽  
Cell Voltage ◽  
Cellular Level ◽  
Whole Cell ◽  
Adult Rat ◽  
Longitudinal Stretch ◽  
Single Channel Currents ◽  
Channel Currents

Mechanoelectric transduction can initiate cardiac arrhythmias. To examine the origins of this effect at the cellular level, we made whole cell voltage-clamp recordings from acutely isolated rat ventricular myocytes under controlled strain. Longitudinal stretch elicited noninactivating inward cationic currents that increased the action potential duration. These stretch-activated currents could be blocked by 100 μM Gd3+ but not by octanol. The current-voltage relationship was nearly linear, with a reversal potential of approximately −6 mV in normal Tyrode solution. Current density varied with sarcomere length (SL) according to I (pA/pF) = 8.3 − 5.0SL (μm). Repeated attempts to record single channel currents from stretch-activated ion channels failed, in accord with the absence of such data from the literature. The inability to record single channel currents may be a result of channels being located on internal membranes such as the T tubules or, possibly, inactivation of the channels by the mechanics of patch formation.

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 Nitric oxide augments single persistent Na+ channel currents via the cGMP/PKG signaling pathway in Kenyon cells isolated from cricket mushroom bodies

2018 ◽  
Vol 120 (2) ◽  
pp. 720-728 ◽  
Author(s):  
Mariko Ikeda ◽  
Masami Yoshino
Keyword(s):  
Nitric Oxide ◽  
Signaling Pathway ◽  
Single Channel ◽  
Signaling Cascade ◽  
Mushroom Bodies ◽  
Na Channel ◽  
No Production ◽  
Kenyon Cells ◽  
Single Channel Currents ◽  
Channel Currents

The nitric oxide (NO)/cyclic GMP signaling pathway has been suggested to be important in the formation of olfactory memory in insects. However, the molecular targets of the NO signaling cascade in the central neurons associated with olfactory learning and memory have not been fully analyzed. In this study, we investigated the effects of NO donors on single voltage-dependent Na+ channels in intrinsic neurons, called Kenyon cells, in the mushroom bodies in the brain of the cricket. Step depolarization on cell-attached patch membranes induces single-channel currents with fast-activating and -inactivating brief openings at the beginning of the voltage steps followed by more persistently recurring brief openings all along the 150-ms pulses. Application of the NO donor S-nitrosoglutathione (GSNO) increased the number of channel openings of both types of single Na+ channels. This excitatory effect of GSNO on the activity of these Na+ channels was diminished by KT5823, an inhibitor of protein kinase G (PKG), indicating an involvement of PKG in the downstream pathway of NO. Application of KT5823 alone decreased the activity of the persistent Na+ channels without significant effects on the fast-inactivating Na+ channels. The membrane-permeable cGMP analog 8Br-cGMP increased the number of channel openings of both types of single Na+ channels, similar to the action of NO. Taken together, these results indicate that NO acts as a critical modulator of both fast-inactivating and persistent Na+ channels and that persistent Na+ channels are constantly upregulated by the endogenous cGMP/PKG signaling cascade. NEW & NOTEWORTHY This study clarified that nitric oxide (NO) increases the activity of both fast-inactivating and persistent Na+ channels via the cGMP/PKG signaling cascade in cricket Kenyon cells. The persistent Na+ channels are also found to be upregulated constantly by endogenous cGMP/PKG signaling. On the basis of the present results and the results of previous studies, we propose a hypothetical model explaining NO production and NO-dependent memory formation in cricket large Kenyon cells.

Anion channels for amino acids in MDCK cells

1992 ◽  
Vol 263 (6) ◽  
pp. C1200-C1207 ◽  
Author(s):  
U. Banderali ◽  
G. Roy
Keyword(s):  
Amino Acids ◽  
Single Channel ◽  
Mdck Cells ◽  
Reversal Potential ◽  
Patch Clamp Technique ◽  
Excised Patches ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Inside Out ◽  
Cytoplasmic Side

Large losses of amino acids by diffusion were previously observed in Madin-Darby canine kidney (MDCK) cells during volume regulation. Also, an outward rectifying anion channel was activated. Because this channel was not selective among anions, it was suggested that it could be permeable to amino acids. Its permeability to aspartate, glutamate, and taurine was studied using the patch-clamp technique in the inside-out configuration. Solutions containing 500 mM aspartate or glutamate were used on the cytoplasmic side of excised patches to detect single-channel currents carried by these anions. Permeability ratios were estimated in two different ways: 1) from the shift in reversal potential of current-voltage curves after anion replacement in the bath solution and 2) from comparisons of amplitudes of single-channel currents carried by tested anions and chloride, respectively. The values of aspartate-to-chloride and glutamate-to-chloride permeability ratios obtained with both methods were quite consistent and were of the order of 0.2 for both amino acids. Taurine in solutions at physiological pH 7.3 is a zwitterionic molecule and bears no net charge. To detect single-channel currents carried by taurine, solutions containing 500 mM taurine at pH 8.2 were used in inside-out experiments. Under these conditions 120 mM of negatively charged taurine was present in the solutions bathing the cytoplasmic side of excised patches. The permeability ratio estimated from the shift in reversal potential was 0.75. These results showed that some of the organic compounds released by cells during regulatory volume decrease could diffuse through this outwardly rectifying anionic channel.

GABA-activated single channel currents in outside-out membrane patches from rat retinal ganglion cells

1989 ◽  
Vol 3 (3) ◽  
pp. 275-279 ◽  
Author(s):  
Stuart A. Lipton
Keyword(s):  
Retinal Ganglion Cells ◽  
Cortical Neurons ◽  
Single Channel ◽  
Ganglion Cells ◽  
Retinal Ganglion ◽  
Whole Cell ◽  
Cell Recording ◽  
Excised Patches ◽  
Patch Configuration ◽  
Single Channel Currents

Abstractγ-aminobutyric acid (GABA) evokes large whole-cell currents in solitary mammalian retinal ganglion cells studied by the patch-clamp method. This evidence suggests that GABA acts directly on the retinal ganglion cells as an inhibitory transmitter as it does elsewhere in the mammalian central nervous system. Here, single-channel recordings of the currents underlying the GABA-induced responses were studied in outside-out patches of cell membrane. In some other preparations, single GABAA channels recorded in the excised patch configuration have been shown to have altered properties in comparison to responses elicited during whole-cell recording. For example, in cortical neurons single GABA-activated channels in excised patches display accelerated desensitization kinetics as well as rapid rundown of the response. Therefore, in retinal ganglion cells, responses generated by GABA in cell-free patches were compared to whole-cell responses. After determining that the responses to GABA in acutely isolated outside-out patches were indeed similar to those of the whole-cell currents in retinal ganglion cells, the unitary conductances were studied. It was determined that these single-channel events resemble those reported in other nervous tissues with 4 elementary conductances of ~10 pS, 19–22 pS, 30–33 pS, and 45–50 pS at 33–35°C.

scholarly journals Effect of N-bromoacetamide on single sodium channel currents in excised membrane patches.

1982 ◽  
Vol 79 (3) ◽  
pp. 333-351 ◽  
Author(s):  
J Patlak ◽  
R Horn
Keyword(s):  
Sodium Channel ◽  
Open Channel ◽  
Single Channel ◽  
Voltage Range ◽  
Cytoplasmic Face ◽  
Before And After ◽  
Excised Patches ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Single Sodium Channel

We have studied the effect of N-bromoacetamide (NBA) on the behavior of single sodium channel currents in excised patches of rat myotube membrane at 10 degree C. Inward sodium currents were activated by voltage steps from holding potentials of about -100 mV to test potentials of -40 mV. The cytoplasmic-face solution was isotonic CsF. Application of NBA or pronase to the cytoplasmic face of the membrane irreversibly removed sodium channel inactivation, as determined by averaged single-channel records. Teh lifetime of the open channel at -40 mV was increased about 10-fold by NBA treatment without affecting the amplitude of single-channel currents. A binomial analysis was used both before and after treatment to determine the number of channels within the excised patch. NBA was shown to have little effect on activation kinetics, as determined by an examination of both the rising phase of averaged currents and measurements f the delay between the start of the pulse and the first channel opening. Our data support a kinetic model of sodium channel activation in which the rate constant leading back from the open state to the last closed state is slower than expected from a strict Hodgkin-Huxley model. The data also suggest that the normal open-channel lifetime is primarily determined by the inactivation process in the voltage range we have examined.

scholarly journals Low pH Potentiates Both Capsaicin Binding and Channel Gating of VR1 Receptors

2003 ◽  
Vol 122 (1) ◽  
pp. 45-61 ◽  
Author(s):  
Sujung Ryu ◽  
Beiying Liu ◽  
Feng Qin
Keyword(s):  
Single Channel ◽  
Channel Gating ◽  
General Mechanism ◽  
Low Concentrations ◽  
Excised Patches ◽  
High Concentrations ◽  
Activation Pathways ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Important Basis

Capsaicin ion channels are highly expressed in peripheral nervous terminals and involved in pain and thermal sensations. One characteristic of the cloned VR1 receptor is its multimodal responses to various types of noxious stimuli. The channel is independently activated by capsaicin and related vanilloids at submicromolar range, by heat above 40°C, and by protons at pH below 6.5. Furthermore, simultaneous applications of two or more stimuli lead to cross sensitization of the receptor, with an apparent increase in the sensitivity to any individual stimulus when applied alone. We studied here the mechanism underlying such cross-sensitization; in particular, between capsaicin and pH, two prototypical stimuli for the channel. By analyzing single-channel currents recorded from excised-patches expressing single recombinant VR1 receptors, we examined the effect of pH on burst properties of capsaicin activation at low concentrations and the effect on gating kinetics at high concentrations. Our results indicate that pH has dual effects on both capsaicin binding and channel gating. Lowering pH enhances the apparent binding affinity of capsaicin, promotes the occurrences of long openings and short closures, and stabilizes at least one of the open conformations of the channel. Our data also demonstrate that capsaicin binding and protonation of the receptor interact allosterically, where the effect of one can be offset by the effect of the other. These results provide important basis to further understand the nature of the activation pathways of the channel evoked by different stimuli as well as the general mechanism underling the cross-sensitization of pain.

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