Glutamate activation of a single NMDA receptor-channel produces a cluster of channel openings.

Activations of the N-methyl-D-aspartate (NMDA) receptor by glutamate were studied in outside-out patches from CA1 cells in rat hippocampal slices. Very low glutamate concentrations (20-100 nM) were used so that individual receptor activations would be well separated. The shut-time distribution contained at least five components, only the longest component being obviously concentration dependent. The three briefest shut-time components had time constants of 56 microseconds, 0.68 ms and 10.1 ms; all of these were independent of glutamate concentration. An individual activation of the receptor therefore produces a long cluster of channel openings that contains longer gaps than have been reported for receptor activations by other fast neurotransmitters. In addition, (i) some activations may contain still longer (mean 78 ms) shut periods generating 'super clusters', and (ii) a significant amount of NMDA current may be carried by prolonged ('high P(open)') periods during which the channel is open for most of the time. Such periods occur intermittently even at these very low glutamate concentrations. It is suggested that the slow time course of the NMDA receptor-mediated synaptic currents may be determined mainly by the channel activation kinetics.     

Some properties of acetylcholine receptors in human cultured myotubes

The distribution and single channel properties of acetylcholine (ACh) receptors in human myotubes grown in tissue culture have been examined. Radioautography of myotubes labelled with [125I]alpha-bungarotoxin showed that ACh receptors are distributed uniformly over the myotube surface at a density of 3.9 +/- 0.5 receptors per square micrometre. Accumulations of ACh receptors (hot spots) were found rarely. The conductance and kinetics of ACh-activated channels were investigated with the patch-clamp technique. Cell-attached membrane patches were used in all experiments. A single channel conductance in the range 40-45 pS was calculated. No sublevels of conductance (substates) of the activated channel were observed. The distribution of channel open-times varied with ACh concentration. With 100 nM ACh, the distribution was best fitted by the sum of two exponentials, whereas with 1 microM ACh a single exponential could be fitted. The mean channel open-time at the myotube resting potential (ca. -70 mV, 22 degrees C) was 8.2 ms. The distribution of channel closed-times was complex at all concentrations of ACh studied (100 nM to 10 microM). With desensitizing doses of ACh (10 microM), channel openings occurred in obvious bursts; each burst usually appeared as part of a 'cluster' of bursts. Both burst duration and mean interval between bursts increased with membrane hyperpolarization. Individual channel open-times and burst durations showed similar voltage dependence (e-fold increase per 80 mV hyperpolarization), whereas both the channel closed-times within a burst and the number of openings per burst were independent of membrane potential.     

The excitatory glutamate-activated channel recorded in cell-attached and excised patches from the membranes of tail,leg and stomach muscles of crayfish

Summary Glutamate activated, excitatory single channel currents were recorded from 5 different muscles of crayfish (Austropotamobius torrentium) from abdomen, legs and stomach. Cell-attached and outside-out excised membrane patches with G-seals were studied. At –70 mV membrane potential and 19 °C, single channel currents activated by 0.5 mM glutamate had an amplitude of –7.6 pA, a mean open time of 0.22 ms and a mean burst length of 0.58 ms. These values did not show significant differences in all muscles investigated. The distributions of open times and of burst durations were described by single exponentials. The distributions of closed times could be fitted only by at least two exponentials. The short component of on average 0.1 ms represented closings within bursts, a longer component of on average 0.9 ms grouping of bursts. Burst durations (but not individual open times) increased with rising glutamate concentration: the relative open time of the channel was approximately proportional to glutamate concentration between 0.1 and 5 mM. The channels described above could not be activated by the glutamate analogues kainate and NMDA, but were about 10 times more sensitive to quisqualate than to glutamate. Quisqualate elicited single channel currents of the same amplitude as those triggered by glutamate. Compared at the same concentrations, channel open times and burst durations were about 4 times longer in quisqualate than in glutamate. A model describing the kinetics of the glutamate-activated excitatory channels is discussed. In addition, a type of Ca-independent, depolarization-activated K⁺-channel is reported.     

Gating of single non-Shaker A-type potassium channels in larval Drosophila neurons

The voltage-dependent gating of transient A2-type potassium channels from primary cultures of larval Drosophila central nervous system neurons was studied using whole-cell and single-channel voltage clamp. A2 channels are genetically distinct from the Shaker A1 channels observed in Drosophila muscle, and differ in single-channel conductance, voltage dependence, and gating kinetics. Single A2 channels were recorded and analyzed at -30, -10, +10, and +30 mV. The channels opened in bursts in response to depolarizing steps, with three to four openings per burst and two to three bursts per 480-ms pulse (2.8-ms burst criterion). Mean open durations were in a range of 2-4 ms and mean burst durations in a range of 9-17 ms. With the exception of the first latency distributions, none of the means of the distributions measured showed a consistent trend with voltage. Macroscopic inactivation of both whole-cell A currents and ensemble average currents of single A2 channels was well fitted by a sum of two exponentials. The fast time constants in different cells were in a range of 9-25 ms, and the slow time constants in a range of 60-140 ms. A six-state kinetic model (three closed, one open, two inactivated states) was tested at four command voltages by fitting frequency histograms of open durations, burst durations, burst closed durations, number of openings per burst, and number of bursts per trace. The model provided good fits to these data, as well as to the ensemble averages. With the exception of the rates leading to initial opening, the transitions in the model were largely independent of voltage.     

Single-channel currents from diethylpyrocarbonate-modified NMDA receptors in cultured rat brain cortical neurons

The role of histidine residues in the function of N-methyl-D-aspartate (NMDA)-activated channels was tested with the histidine-modifying reagent diethylpyrocarbonate (DEP) applied to cells and membrane patches from rat brain cortical neurons in culture. Channels in excised outside-out patches that were treated with 3 mM DEP for 15-30 s (pH 6.5) showed an average 3.4-fold potentiation in steady state open probability when exposed to NMDA and glycine. Analysis of the underlying alterations in channel gating revealed no changes in the numbers of kinetic states: distributions of open intervals were fitted with three exponential components, and four components described the shut intervals, in both control and DEP-modified channels. However, the distribution of shut intervals was obviously different after DEP treatment, consistent with the single-channel current record. After modification, the proportion of long shut states was decreased while the time constants were largely unaffected. Burst kinetics reflected these effects with an increase in the average number of openings/burst from 1.5 (control) to 2.2 (DEP), and a decrease in the average interburst interval from 54.1 to 38.2 ms. These effects were most likely due to histidine modification because other reagents (n- acetylimidazole and 2,4,6-trinitrobenzene 1-sulfonic acid) that are specific for residues other than histidine failed to reproduce the effects of DEP, whereas hydroxylamine could restore channel open probability to control levels. In contrast to these effects on channel gating, DEP had no effect on average single-channel conductance or reversal potential under bi-ionic (Na+:Cs+) conditions. Inhibition by zinc was also unaffected by DEP. We propose a channel gating model in which transitions between single- and multi-opening burst modes give rise to the channel activity observed under steady state conditions. When adjusted to account for the effects of DEP, this model suggests that one or more extracellular histidine residues involved in channel gating are associated with a single kinetic state.     

Kinetic diversity of Na+ channel bursts in frog skeletal muscle

Individual Na+ channels of dissociated frog skeletal muscle cells at 10 degrees C fail to inactivate in 0.02% of depolarizing pulses, thus producing bursts of openings lasting hundreds of milliseconds. We present here a kinetic analysis of 87 such bursts that were recorded in multi-channel patches at four pulse potentials. We used standard dwell-time histograms as well as fluctuation analysis to analyze the gating kinetics of the bursting channels. Since each burst contained only 75-150 openings, detailed characterization of the kinetics from single bursts was not possible. Nevertheless, at this low kinetic resolution, the open and closed times could be well fitted by single exponentials (or Lorentzians for the power spectra). The best estimates of both the open and closed time constants produced by either technique were much more broadly dispersed then expected from experimental or analytical variability, with values varying by as much as an order of magnitude. Furthermore, the values of the open and closed time constants were not significantly correlated with one another from burst to burst. The bursts thus expressed diverse kinetic behaviors, all of which appear to be manifestations of a single type of Na+ channel. Although the opening and closing rates were dispersed, their average values were close to those of alpha m and 2 beta m derived from fits to the early transient Na+ currents over the same voltage range. We propose a model in which the channel has both primary states (e.g., open, closed, and inactivated), as well as "modes" that are associated with independent alterations in the rate constants for transition between each of these primary states.     

Two modes of gating during late Na+ channel currents in frog sartorius muscle

Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.     

Regulation of cytoplasmic pH of cultured bovine corneal endothelial cells in the absence and presence of bicarbonate

Summary Single sodium-channel currents were measured in neuroblastoma cells after inhibition of inactivation by chloramine-T (CHL-T), sea anemone toxin II (ATX-II) and scorpion toxin (SCT). The decaying phase of the averaged single-channel currents recorded with 90-msec pulses in cell-attached patches was clearly slower than that of the unmodified channels, suggesting inhibition of macroscopic inactivation. Each substance caused repetitive openings and a moderate increase in the channel open time. AtV _m =RP+20 mV andT=12°C, the mean channel open times were 1.4, 1.6 and 1.8 msec for CHL-T, ATX-II and SCT, respectively, as opposed to 1.07 msec for native channels. Open-time histograms could be best fitted by the sum of two exponentials. The time constants of the fits were similar for histograms constructed from single openings and from openings during bursts. This suggests that the population of channels is homogeneous and that in bursts the same open conformations of channels occur as in single openings. Mean burst durations for bursts consisting of more than one opening atV _m =RP+20 mV were 4.9, 5.8 and 6.1 msec for CHL-T, ATX-II and SCT, respectively. Burst open-time histograms constructed from two or three openings were fitted by the gamma function. The different time constants of the fits obtained for ATX-II and SCT suggested multiple open conformations of channels for openings of bursts. However, significantly different open-time histograms constructed from the first, second and third openings of bursts could not be obtained systematically. A positive correlation was found for the dwell time of the first and the second, as well as for the second and the third opening of bursts with each substance, but a negative one for the dwell time of an opening and the neighboring closing of bursts with ATX-II. The results suggest a model with multiple open and inactivated states. In this model the inactivated states are weakly absorbing.     

海马神经元乙酰胆碱激活通道在不同培养期的功能特性

用膜片箝技术对不同培养期的新生大鼠海马神经元上乙酰胆碱受体单通道特性进行了研究,结果表明不同培养期ＡＣｈ激活通道的电学特性不同。培养早期（１－２ｄ）,２０ｐｓ通道占优势,开放以单个短开放事件为主,平均开放时间小于２ｍｓ．培养后期（１８－２１ｄ）３１,ｐＳ通道为主,开放随膜片的不同可分成两类,即单个短开放（时间常数为０．３５ｍｓ和１．２９ｍｓ）和簇状开放（时间常数为１．１５ｍｓ和９．６ｍｓ）,同时也     

Rapid activation, desensitization, and resensitization of synaptic channels of crayfish muscle after glutamate pulses.

Completely desensitizing excitatory channels were activated in outside-out patches of crayfish muscle membrane by applying glutamate pulses with switching times of approximately 0.2 ms for concentration changes. Channels were almost completely activated with 10 mM glutamate. Maximum activation was reached within 0.4 ms with greater than or equal to 1 mM glutamate. Channel open probability decayed with a time constant of desensitization of 2 ms with 10 mM glutamate and more rapidly at lower glutamate concentrations. The rate of beginnings of bursts (average number of beginnings of bursts per time bin) decayed even faster but approximately in proportion to the glutamate concentration. The dose-response curve for the channel open probability and for the rate of bursts had a maximum double-logarithmic slope of 5.1 and 4.2, respectively. Channels desensitized completely without opening at very low or slowly rising glutamate concentrations. Desensitization thus originates from a closed channel state. Resensitization was tested by pairs of completely desensitizing glutamate pulses. Sensitivity to the second pulse returned rapidly at pulse intervals between 1 and 2 ms and was almost complete with an interval of 3 ms. Schemes of channel activation by up to five glutamate binding steps, with desensitization by glutamate binding from closed states, are discussed. At high agonist concentrations bursts are predominantly terminated by desensitization. Quantal currents are generated by pulses of greater than 1 mM glutamate, and their decay is determined by the duration of presence of glutamate and possibly by desensitization.     

Activation kinetics reveal the number of glutamate and glycine binding sites on the N-methyl-D-aspartate receptor.

The activation kinetics of N-methyl-D-aspartate (NMDA) channels in outside-out patches from cultured hippocampal neurons were analyzed to determine the number of glutamate and glycine binding sites per channel. Following rapid steps into high concentrations of glutamate, the activation time course was concentration-independent and limited by transitions between the shut, but fully liganded state and the open state. At lower concentrations, ligand binding was rate-limiting. The resulting sigmoidal activation time course was best fitted by a kinetic model with two glutamate binding sites. Glycine concentration jumps in the continuous presence of glutamate were also best fitted with a two-site model. Agonist and co-agonist binding were better described by an independent, rather than a sequential model. We suggest that the NMDA receptor is at least a tetramer containing four ligand binding subunits, assuming a single binding site per subunit.     

Properties of Acetylcholine Receptor Ion Channels in the Acutely Dissociated Neurons of the Marginal Division in the Rat Striatum

Cell-attached mode of patch clamp technique was employed to investigate the properties of acetylcholine (ACh)-induced ion channels in acutely dissociated neurons from the marginal division (MrD) of rat striatum. Two types of conductance states (25 pS and 60 pS) were recorded. The 25 pS channel (more than 80%) was the main type in the neurons of MrD and was described here. The amplitudes of inward currents increased with hyperpolorization and the reversing potential was about 0 mV. Both single short opening and long burst openings were observed in MrD neurons. Two time constants of these two kinds of ion channels are 0.29 ms, 1.84 ms and 1.96 ms, 18.24 ms, respectively. Average close time can be fitted with two exponential functions, the two time constants are 1.7 ms and 54 ms. Probability of channel opening is about 0.012 and no voltage-dependence was found. The properties of reversing potential, voltage-independence and the form of agonist to the ion channels indicated that the recorded channel currents flow through AChR channels. The mAChR is involved in slow synaptic transmission and Ach can not induce the opening of mAChR ion channel. The binding site of ACh to AChR and the nAChR ion channel are the same protein, ACh can only activate nAChR ion channel directly. Therefore, the recorded ion channels in the present study are nAChR ion channels. The results suggest that nAChR ion channels exist in the neurons of MrD and the MrD probably is involved in learning and memory mechanism of the brain.     

Kinetic properties of single sodium channels in rat heart and rat brain

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.     

Mutations within the P-loop of Kir6.2 modulate the intraburst kinetics of the ATP-sensitive potassium channel

The ATP-sensitive potassium (K(ATP)) 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 K(ATP) 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 (tau(o)) and the short closed time (tauC1) 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 tau(o), shorter tauC1, 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 K(ATP) 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.     

Fine gating properties of channels responsible for persistent sodium current generation in entorhinal cortex neurons

The gating properties of channels responsible for the generation of persistent Na(+) current (I(NaP)) in entorhinal cortex layer II principal neurons were investigated by performing cell-attached, patch-clamp experiments in acutely isolated cells. Voltage-gated Na(+)-channel activity was routinely elicited by applying 500-ms depolarizing test pulses positive to -60 mV from a holding potential of -100 mV. The channel activity underlying I(NaP) consisted of prolonged and frequently delayed bursts during which repetitive openings were separated by short closings. The mean duration of openings within bursts was strongly voltage dependent, and increased by e times per every approximately 12 mV of depolarization. On the other hand, intraburst closed times showed no major voltage dependence. The mean duration of burst events was also relatively voltage insensitive. The analysis of burst-duration frequency distribution returned two major, relatively voltage-independent time constants of approximately 28 and approximately 190 ms. The probability of burst openings to occur also appeared largely voltage independent. Because of the above "persistent" Na(+)-channel properties, the voltage dependence of the conductance underlying whole-cell I(NaP) turned out to be largely the consequence of the pronounced voltage dependence of intraburst open times. On the other hand, some kinetic properties of the macroscopic I(NaP), and in particular the fast and intermediate I(NaP)-decay components observed during step depolarizations, were found to largely reflect mean burst duration of the underlying channel openings. A further I(NaP) decay process, namely slow inactivation, was paralleled instead by a progressive increase of interburst closed times during the application of long-lasting (i.e., 20 s) depolarizing pulses. In addition, long-lasting depolarizations also promoted a channel gating modality characterized by shorter burst durations than normally seen using 500-ms test pulses, with a predominant burst-duration time constant of approximately 5-6 ms. The above data, therefore, provide a detailed picture of the single-channel bases of I(NaP) voltage-dependent and kinetic properties in entorhinal cortex layer II neurons.     

Modal gating behavior of cardiac sodium channels in cell-free membrane patches.

Single voltage-activated Na+ channel currents were obtained from membrane patches of isolated ventricular cells of guinea pig hearts. The currents were compared when measured from cell-attached patches and from the same patch but at least 20 minutes after manual excision. The averaged currents showed a distinctly delayed decay in the excised patches due to the appearance of long lasting openings or bursts of openings. In contrast to control patches, the open time distribution in excised patches requires at least two exponentials. A short mean open time was voltage independent for cell-attached patches (0.38 ms +/- 0.07 ms between -60 and -20 mV, 6 cell-attached patches; and 0.41 +/- 0.1 ms, 7 excised patches). The long mean open time found in excised patches was clearly voltage dependent and increased from 0.48 +/- 0.14 ms (-80 mV) to 2.87 +/- 0.35 ms (-20 mV, regression coefficient +0.88, 7 patches). Sweeps with long openings appeared in clusters. The clustering of records with long openings, short openings, or without openings (nulls) was quantified by a runs analysis which showed a highly significant nonrandom ordering. The results show that in excised patches inactivation is temporally hibernating.     

Opentime heterogeneity during bursting of sodium channels in frog skeletal muscle.

Single voltage-activated Na+ channel currents were obtained from membrane patches on internally dialyzed skeletal muscle segments of adult frogs. The high channel density in these membranes permitted frequent observation of the "bursting mode" of individual Na+ channels during 400-ms records. We examined the opentimes within and between bursts on individual membrane patches. We used a new nonparametric statistical procedure to test for heterogeneity in the opentime distributions. We found that although 80% of all bursts consisted of opentimes drawn from a single distribution, the opentime distribution varied significantly from burst to burst. Significant heterogeneity was also detected within the remaining 20% of individual bursts. Our results indicate that the gating kinetics of individual Na+ channels are heterogeneous, and that they may occasionally change in a single channel.     

Autoreceptor Regulation of Glutamate and Aspartate Release from Slices of the Hippocampal CA1 Area

Slices of hippocampal area CA1 were employed to test the hypothesis that the release of glutamate and aspartate is regulated by the activation of excitatory amino acid autoreceptors. In the absence of added Mg2+, N-methyl-D-aspartate (NMDA)-receptor antagonists depressed the release of glutamate, aspartate, and gamma-aminobutyrate evoked by 50 mM K+. Conversely, the agonist NMDA selectively enhanced the release of aspartate. The latter action was observed, however, only when the K+ stimulus was reduced to 30 mM. Actions of the competitive antagonists 3-[(+/- )-2-carboxypiperazin-4-yl]-propyl-l-phosphonic acid (CPP) and D-2-amino-5-phosphonovalerate (D-AP5) differed, in that the addition of either 1.2 mM Mg2+ or 0.1 microM tetrodotoxin to the superfusion medium abolished the depressant effect of CPP without diminishing the effect of D-AP5. These results suggest that the activation of NMDA receptors by endogenous glutamate and aspartate enhances the subsequent release of these amino acids. The cellular mechanism may involve Ca2+ influx through presynaptic NMDA receptor channels or liberation of a diffusible neuromodulator linked to the activation of postsynaptic NMDA receptors. (RS)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, a selective quisqualate receptor agonist, and kainate, an agonist active at both kainate and quisqualate receptors, selectively depressed the K(+)-evoked release of aspartate. Conversely, 6-cyano-7-nitro-quinoxaline-2,3-dione, an antagonist active at both quisqualate and kainate receptors, selectively enhanced aspartate release. These results suggest that glutamate can negatively modulate the release of aspartate by activating autoreceptors of the quisqualate, and possibly also of the kainate, type. Thus, the activation of excitatory amino acid receptors has both presynaptic and postsynaptic effects.     

Cell responses to single pheromone molecules may reflect the activation kinetics of olfactory receptor molecules

Olfactory receptor cells of the silkmoth Bombyx mori respond to single pheromone molecules with "elementary" electrical events that appear as discrete "bumps" a few milliseconds in duration, or bursts of bumps. As revealed by simulation, one bump may result from a series of random openings of one or several ion channels, producing an average inward membrane current of 1.5 pA. The distributions of durations of bumps and of gaps between bumps in a burst can be fitted by single exponentials with time constants of 10.2 ms and 40.5 ms, respectively. The distribution of burst durations is a sum of two exponentials; the number of bumps per burst obeyed a geometric distribution (mean 3.2 bumps per burst). Accordingly the elementary events could reflect transitions among three states of the pheromone receptor molecule: the vacant receptor (state 1), the pheromone-receptor complex (state 2), and the activated complex (state 3). The calculated rate constants of the transitions between states are k(21)=7.7 s(-1), k(23)=16.8 s(-1), and k(32)=98 s(-1).     

Kinetics of P2X7 receptor-operated single channels currents

Human P2X7 receptors were expressed in Xenopus laevis oocytes and single channels were recorded using the patch-clamp technique in the outside-out configuration. ATP4- evoked two types of P2X7 receptor-mediated single channel currents characterized by short-lived and long-lived openings. The short- and long-lasting open states had mean open times of approximately 5 and approximately 20 ms and slope conductances near -60 mV of 9 and 13 pS, respectively. The open probabilities of the short and long openings were strongly [ATP4-]-dependent with EC50 values of approximately 0.3 mM and approximately 0.1 mM ATP4-, respectively. The channel kinetics did not change significantly during sustained P2X7 receptor activation for several minutes, as was also observed in recordings in the cell-attached patch-clamp configuration. Activation and deactivation of the short openings followed exponential time courses with time constants in the range of 20 ms, and displayed a shallow [ATP4-] dependence of the activation process. The kinetics of the short channel openings at negative membrane potentials fitted well to a linear C-C-C-O model with two ATP4- binding steps at equal binding sites with a dissociation constant Kd of 139 microM.