scholarly journals Single Channel Properties of P2X2 Purinoceptors

1999 ◽  
Vol 113 (5) ◽  
pp. 695-720 ◽  
Author(s):  
Shinghua Ding ◽  
Frederick Sachs
Keyword(s):  
Conformational Changes ◽  
Single Channel ◽  
Excess Noise ◽  
Hill Coefficient ◽  
Inward Rectification ◽  
Channel Current ◽  
Single Channel Current ◽  
The Mean ◽  
Single Channel Currents ◽  
Channel Properties

The single channel properties of cloned P2X2 purinoceptors expressed in human embryonic kidney (HEK) 293 cells and Xenopus oocytes were studied in outside-out patches. The mean single channel current–voltage relationship exhibited inward rectification in symmetric solutions with a chord conductance of ∼30 pS at −100 mV in 145 mM NaCl. The channel open state exhibited fast flickering with significant power beyond 10 kHz. Conformational changes, not ionic blockade, appeared responsible for the flickering. The equilibrium constant of Na+ binding in the pore was ∼150 mM at 0 mV and voltage dependent. The binding site appeared to be ∼0.2 of the electrical distance from the extracellular surface. The mean channel current and the excess noise had the selectivity: K+ > Rb+ > Cs+ > Na+ > Li+. ATP increased the probability of being open (Po) to a maximum of 0.6 with an EC50 of 11.2 μM and a Hill coefficient of 2.3. Lowering extracellular pH enhanced the apparent affinity of the channel for ATP with a pKa of ∼7.9, but did not cause a proton block of the open channel. High pH slowed the rise time to steps of ATP without affecting the fall time. The mean single channel amplitude was independent of pH, but the excess noise increased with decreasing pH. Kinetic analysis showed that ATP shortened the mean closed time but did not affect the mean open time. Maximum likelihood kinetic fitting of idealized single channel currents at different ATP concentrations produced a model with four sequential closed states (three binding steps) branching to two open states that converged on a final closed state. The ATP association rates increased with the sequential binding of ATP showing that the binding sites are not independent, but positively cooperative. Partially liganded channels do not appear to open. The predicted Po vs. ATP concentration closely matches the single channel current dose–response curve.

scholarly journals Single-Channel Properties of Recombinant Acid-Sensitive Ion Channels Formed by the Subunits Asic2 and Asic3 from Dorsal Root Ganglion Neurons Expressed in Xenopus Oocytes

2001 ◽  
Vol 117 (6) ◽  
pp. 563-572 ◽  
Author(s):  
Ping Zhang ◽  
Cecilia M. Canessa
Keyword(s):  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Dorsal Root Ganglion Neurons ◽  
Channel Current ◽  
Single Channel Current ◽  
The Mean ◽  
Ganglion Neurons ◽  
Channel Properties

The acid-sensitive ion channels known as ASIC are gated by external protons. A set of these channels is expressed in dorsal root ganglion neurons where they may participate in the transduction of mechanical and nociceptive stimuli. Here, we have examined the single-channel properties of channels formed by the subunits ASIC2 and ASIC3 expressed in Xenopus oocytes using outside-out patches. The mean single-channel current-voltage relationship is linear with a slope conductance of 18 pS between −80 and −40 mV in 150 mM Na+ outside and 150 mM K+ inside the patch pipet. The selectivity for monovalent cations has the sequence Na+ > Li+ > K+. Divalent cations such as Ca2+ do not permeate, but instead block the channel when applied to the extracellular side. External protons increase the probability of channels being open to a maximum of 0.8 with an EC50 of 16 ± 4 μM and a Hill coefficient of 2.7 ± 0.3, whereas the mean single-channel current amplitude is independent of external pH. Analysis of the kinetics of single channels indicates the presence of at least four modes of activity (Mod1 to Mod4) in addition to an inactivated state. Three of the modes exhibit distinct kinetics, and can be unambiguously identified on the basis of open probability (PoMod1 = 0.5 ± 0.05; PoMod2 > 0.9 ± 0.05; PoMod3 < 0.1). Mode 4, which has a Po in the range of 0.5–0.8, may constitute a distinct mode or alternatively, it represents transitions between the other three modes of activity. Increasing [H+]o increases the frequency of entering the modes with high Po (modes 1, 2, and 4) and the time the channel spends in the modes with high activity.

scholarly journals Stoichiometry of Recombinant N-Methyl-d-Aspartate Receptor Channels Inferred from Single-channel Current Patterns

1997 ◽  
Vol 110 (5) ◽  
pp. 485-502 ◽  
Author(s):  
Louis S. Premkumar ◽  
Anthony Auerbach
Keyword(s):  
Single Channel ◽  
Wild Type ◽  
Current Pattern ◽  
Channel Current ◽  
Single Channel Current ◽  
Aspartate Receptor ◽  
Single Channel Currents ◽  
Nr2 Subunits ◽  
Channel Currents ◽  
Straightforward Interpretation

Single-channel currents were recorded from mouse NR1-NR2B (ζ-ε2) receptors containing mixtures of wild-type and mutant subunits expressed in Xenopus oocytes. Mutant subunits had an asparagine-to-glutamine (N-to-Q) mutation at the N0 site of the M2 segment (NR1:598, NR2B:589). Receptors with pure N or Q NR1 and NR2 subunits generated single-channel currents with distinctive current patterns. Based on main and sublevel amplitudes, occupancy probabilities, and lifetimes, four patterns of current were identified, corresponding to receptors with the following subunit compositions (NR1/NR2): N/N, N/Q, Q/N, and Q/Q. Only one current pattern was apparent for each composition. When a mixture of N and Q NR2 subunits was coexpressed with pure mutant NR1 subunits, three single-channel current patterns were apparent. One pattern was the same as Q/Q receptors and another was the same as Q/N receptors. The third, novel pattern presumably arose from hybrid receptors having both N and Q NR2 subunits. When a mixture of N and Q NR1 subunits was coexpressed with pure mutant NR2 subunits, six single-channel current patterns were apparent. One pattern was the same as Q/Q receptors and another was the same as N/Q receptors. The four novel patterns presumably arose from hybrid receptors having both N and Q NR1 subunits. The relative frequency of NR1 hybrid receptor current patterns depended on the relative amounts of Q and N subunits that were injected into the oocytes. The number of hybrid receptor patterns suggests that there are two NR2 subunits per receptor and is consistent with either three or five NR1 subunits per receptor, depending on whether or not the order of mutant and wild-type subunits influences the current pattern. When considered in relation to other studies, the most straightforward interpretation of the results is that N-methyl-d-aspartate receptors are pentamers composed of three NR1 and two NR2 subunits.

scholarly journals Endogenous Protease Activation of ENaC

2005 ◽  
Vol 126 (4) ◽  
pp. 339-352 ◽  
Author(s):  
Adedotun Adebamiro ◽  
Yi Cheng ◽  
John P. Johnson ◽  
Robert J. Bridges
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Open Channels ◽  
Na Channel ◽  
Fluctuation Analysis ◽  
Protease Inhibition ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Channel Properties

Endogenous serine proteases have been reported to control the reabsorption of Na+ by kidney- and lung-derived epithelial cells via stimulation of electrogenic Na+ transport mediated by the epithelial Na+ channel (ENaC). In this study we investigated the effects of aprotinin on ENaC single channel properties using transepithelial fluctuation analysis in the amphibian kidney epithelium, A6. Aprotinin caused a time- and concentration-dependent inhibition (84 ± 10.5%) in the amiloride-sensitive sodium transport (INa) with a time constant of 18 min and half maximal inhibition constant of 1 μM. Analysis of amiloride analogue blocker–induced fluctuations in INa showed linear rate–concentration plots with identical blocker on and off rates in control and aprotinin-inhibited conditions. Verification of open-block kinetics allowed for the use of a pulse protocol method (Helman, S.I., X. Liu, K. Baldwin, B.L. Blazer-Yost, and W.J. Els. 1998. Am. J. Physiol. 274:C947–C957) to study the same cells under different conditions as well as the reversibility of the aprotinin effect on single channel properties. Aprotinin caused reversible changes in all three single channel properties but only the change in the number of open channels was consistent with the inhibition of INa. A 50% decrease in INa was accompanied by 50% increases in the single channel current and open probability but an 80% decrease in the number of open channels. Washout of aprotinin led to a time-dependent restoration of INa as well as the single channel properties to the control, pre-aprotinin, values. We conclude that protease regulation of INa is mediated by changes in the number of open channels in the apical membrane. The increase in the single channel current caused by protease inhibition can be explained by a hyperpolarization of the apical membrane potential as active Na+ channels are retrieved. The paradoxical increase in channel open probability caused by protease inhibition will require further investigation but does suggest a potential compensatory regulatory mechanism to maintain INa at some minimal threshold value.

Pathways of HERG inactivation

1999 ◽  
Vol 277 (1) ◽  
pp. H199-H210 ◽  
Author(s):  
Johann Kiehn ◽  
Antonio E. Lacerda ◽  
Arthur M. Brown
Keyword(s):  
Xenopus Oocytes ◽  
Single Channel ◽  
Inward Rectification ◽  
Single Channels ◽  
Channel Current ◽  
Cardiac Action ◽  
Current Voltage ◽  
Inwardly Rectifying ◽  
Channel Properties ◽  
Current Voltage Relation

The rapid, repolarizing K+ current in cardiomyocytes ( I Kr) has unique inwardly rectifying properties that contribute importantly to the downstroke of the cardiac action potential. The human ether-à-go-go-related gene ( HERG) expresses a macroscopic current virtually identical to I Kr, but a description of the single-channel properties that cause rectification is lacking. For this reason we measured single-channel and macropatch currents heterologously expressed by HERG in Xenopus oocytes. Our experiments had two main findings. First, the single-channel current-voltage relation showed inward rectification, and conductance was 9.7 pS at −100 mV and 3.9 pS at 100 mV when measured in symmetrical 100 mM K+ solutions. Second, single channels frequently showed no openings during depolarization but nevertheless revealed bursts of openings during repolarization. This type of gating may explain the inward rectification of HERG currents. To test this hypothesis, we used a three-closed state kinetics model and obtained rate constants from fits to macropatch data. Results from the model are consistent with rapid inactivation from closed states as a significant source of HERG rectification.

scholarly journals Single-Channel Properties of IKs Potassium Channels

1998 ◽  
Vol 112 (6) ◽  
pp. 665-678 ◽  
Author(s):  
Youshan Yang ◽  
Fred J. Sigworth
Keyword(s):  
Single Channel ◽  
Noise Analysis ◽  
Potassium Conductance ◽  
Power Spectra ◽  
Fluctuation Analysis ◽  
Channel Current ◽  
Single Channel Current ◽  
Voltage Dependent ◽  
External Potassium ◽  
Channel Properties

Expressed in Xenopus oocytes, KvLQT1 channel subunits yield a small, rapidly activating, voltage- dependent potassium conductance. When coexpressed with the minK gene product, a slowly activating and much larger potassium current results. Using fluctuation analysis and single-channel recordings, we have studied the currents formed by human KvLQT1 subunits alone and in conjunction with human or rat minK subunits. With low external K+, the single-channel conductances of these three channel types are estimated to be 0.7, 4.5, and 6.5 pS, respectively, based on noise analysis at 20 kHz bandwidth of currents at +50 mV. Power spectra computed over the range 0.1 Hz–20 kHz show a weak frequency dependence, consistent with current interruptions occurring on a broad range of time scales. The broad spectrum causes the apparent single-channel current value to depend on the bandwidth of the recording, and is mirrored in very “flickery” single-channel events of the channels from coexpressed KvLQT1 and human minK subunits. The increase in macroscopic current due to the presence of the minK subunit is accounted for by the increased apparent single-channel conductance it confers on the expressed channels. The rat minK subunit also confers the property that the outward single-channel current is increased by external potassium ions.

Na(+)-activated nonselective cation channels in primary olfactory neurons

1995 ◽  
Vol 73 (5) ◽  
pp. 1774-1781 ◽  
Author(s):  
A. B. Zhainazarov ◽  
B. W. Ache
Keyword(s):  
Neuronal Excitability ◽  
Single Channel ◽  
Cation Channel ◽  
Hill Coefficient ◽  
Inward Rectification ◽  
Dependent Manner ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Channel Current ◽  
Current Voltage

1. Excised inside-out patch recordings were used to describe a novel cation channel from cultured lobster olfactory receptor neurons that is activated by [Na+]i. 2. The channel is reversibly activated by intracellular Na+ as low as 5 mM. The half-effect concentration of intracellular Na+ is approximately 60 mM at -60 mV. The dependence of the channel open probability on [Na+]i is sigmoidal with a Hill coefficient of 3.1, indicating that more than one Na+ must bind to activate the channel. 3. The channel is equally permeable to Na+, K+, and Li+. In symmetrical 210 mM Na+, the open channel current-voltage relationship shows slight inward rectification at positive potentials. The slope conductance of the channel is 107 pS between -90 and 0 mV. 4. Although the channel is not activated by voltage in the absence of intracellular Na+, the gating of the channel is dependent on voltage as well as [Na+]i and [Na+]o. 5. Both intracellular Ca2+ and Mg2+ reversibly affect channel activity in a concentration-dependent manner starting at 1 microM. Ca2+ decreases both the open probability and the single channel amplitude, whereas Mg2+ decreases the open probability but has no effect on the single channel amplitude. Ba2+ (5 mM), but not 20 mM Cs+ and 100 microM amiloride, reversibly block the channel. 6. We speculate that this novel cation channel regulates neuronal excitability by accentuating the rate and/or the magnitude of depolarization of the cell to odors.

Number of KCa Channels Underlying Spontaneous Miniature Outward Currents (SMOCs) in Mudpuppy Cardiac Neurons

2001 ◽  
Vol 85 (1) ◽  
pp. 54-60 ◽  
Author(s):  
Fabiana S. Scornik ◽  
Laura A. Merriam ◽  
Rodney L. Parsons
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Current Amplitude ◽  
Channel Current ◽  
Single Channel Current ◽  
Outward Currents ◽  
Intracellular Ca ◽  
Single Channel Currents ◽  
Channel Currents

Spontaneous miniature outward currents (SMOCs) in parasympathetic neurons from mudpuppy cardiac ganglia are caused by activation of TEA- and iberiotoxin-sensitive, Ca2+-dependent K+(BK) channels. Previously we reported that SMOCs are activated by Ca2+-induced Ca2+ release (CICR) from caffeine- and ryanodine-sensitive intracellular Ca2+ stores. In the present study, we analyzed the single channel currents that contribute to SMOC generation in mudpuppy cardiac neurons. The slope conductance of BK channels, determined from the I-V relationship of single-channel currents recorded with cell-attached patches in physiological K+ concentrations, was 84 pS. The evidence supporting the identity of this channel as the channel involved in SMOC generation was its sensitivity to internal Ca2+, external TEA, and caffeine. In cell-attached patch recordings, 166 μM TEA applied in the pipette reduced single-channel current amplitude by 32%, and bath-applied caffeine increased BK channel activity. The ratio between the averaged SMOC amplitude and the single-channel current amplitude was used to estimate the average number of channels involved in SMOC generation. The estimated number of channels involved in generation of an averaged SMOC ranged from 18 to 23 channels. We also determined that the Po of the BK channels at the peak of a SMOC remains constant at voltages more positive than −20 mV, suggesting that the transient rise in intracellular Ca2+from ryanodine-sensitive intracellular stores in the vicinity of the BK channel reached concentrations most likely exceeding 40 μM.

Effect of Prozac on whole cell ionic currents in lens and corneal epithelia

1995 ◽  
Vol 269 (1) ◽  
pp. C250-C256 ◽  
Author(s):  
J. L. Rae ◽  
A. Rich ◽  
A. C. Zamudio ◽  
O. A. Candia
Keyword(s):  
Potassium Channels ◽  
Single Channel ◽  
Ionic Currents ◽  
Current Amplitude ◽  
Human Lens ◽  
Delayed Rectifier ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Ionic Conductances

Prozac (fluoxetine), a compound used therapeutically in humans to combat depression, has substantial effects on ionic conductances in rabbit corneal epithelial cells and in cultured human lens epithelium. In corneal epithelium, it reduces the current due to the large-conductance potassium channels that dominate this preparation. Its effects seem largely to decrease the open probability while leaving the single-channel current amplitude unaltered. In cultured human epithelium, currents from calcium-activated potassium channels and inward rectifiers are unaffected by Prozac. Delayed-rectifier potassium currents are reduced by Prozac in a complicated way that involves both gating and single-channel current amplitude. Fast tetrodotoxin-blockable sodium currents are also decreased by Prozac in this preparation. For all of these ion conductance effects, Prozac concentrations of 10(-5) to 10(-4) M are required. Whereas these levels are 10- to 100-fold higher than the plasma levels achieved in therapeutic use in humans, they are comparable to or less than levels needed for many other blockers of the ionic conductances studied here.

Properties and distribution of single voltage-gated calcium channels in adult hippocampal neurons

1990 ◽  
Vol 64 (1) ◽  
pp. 91-104 ◽  
Author(s):  
R. E. Fisher ◽  
R. Gray ◽  
D. Johnston
Keyword(s):  
Guinea Pig ◽  
Calcium Channels ◽  
Single Channel ◽  
Cell Types ◽  
Voltage Gated ◽  
Channel Current ◽  
Single Channel Current ◽  
Voltage Gated Calcium Channels ◽  
Large Conductance Channel ◽  
Small Conductance

1. The properties of single voltage-gated calcium channels were investigated in acutely exposed CA3 and CA1 pyramidal neurons and granule cells of area dentata in the adult guinea pig hippocampal formation. 2. Guinea pig hippocampal slices were prepared in a conventional manner, then treated with proteolytic enzymes and gently shaken to expose the somata of the three cell types studied. Standard patch-clamp techniques were used to record current flow through calcium channels in cell-attached membrane patches with isotonic barium as the charge carrier. 3. Single-channel current amplitudes were measured at different membrane potentials. Single-channel current-voltage plots were constructed and single-channel slope conductances were found to fall into three classes. These were (approximately) 8, 14, and 25 pS, and were observed in all three cell types. 4. The three groups of channels differed from each other in voltage dependence of activation: from a holding potential of -80, the small-conductance channel began to activate at about -40 to -30 mV, the medium-conductance channel at about -20 mV, and the large-conductance channel at approximately 0 mV. 5. Ensemble averages of single-channel currents during voltage steps revealed differences in voltage-dependent inactivation. The small-conductance channel inactivated completely within approximately 50 ms during steps from -80 to -10 mV or more positive. Steps to less positive potentials resulted in less inactivation. The medium-conductance channel displayed variable inactivation during steps from -80 to 0 mV. Inactivation of this channel during a 160-ms step ranged from virtually zero to approximately 100%. The large-conductance channel displayed no significant inactivation during steps as long as 400 ms. 6. The large-conductance channel was strikingly affected by the dihydropyridine agonist Bay K8644 (0.5-2.0 microM), resulting in a high probability of channel opening, prolonged openings, and an apparent increase in the number of channels available for activation. The medium and small-conductance channels were not noticeably affected by the drug. 7. The large-conductance channel could be induced to open at very negative membrane potentials by holding the patch for several seconds at 20 or 30 mV and stepping to -30 or -40 mV. This process was enhanced by Bay K8644, resulting in prolonged openings at potentials as negative as -100 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

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