Single-channel evidence for glycine and NMDA requirement in NMDA receptor activation.

N-Methyl-D-aspartate (NMDA) receptor dose-response relationships that are based on macroscopic currents suggest that NMDA and a different agonist molecule, glycine, must together activate the channel. Since single-channel recordings have a much higher resolution than whole-cell currents, they provide a highly sensitive test for the absolute requirement of NMDA channel opening for glycine. Rapid application of 10-300 microM NMDA to outside-out patches from cultured cortical neurons evoked substantial single-channel activity in the absence of added glycine. However, in the presence of a high affinity and highly selective glycine-site antagonist, 5,7-dichlorokynurenate (DCK), NMDA failed to evoke any openings on its own. Channel openings could not be produced by saturating concentrations of NMDA (up to 1 mM) but were evoked when glycine was added to the test solution. Glycine alone (up to 100 microM) was similarly ineffective in the continuous presence of D(-)-2-amino-5-phosphonovaleric acid (D-APV), an NMDA-site antagonist. Reversal of antagonist blockade by the appropriate ligand (glycine or NMDA) and the normal appearance and duration of channel openings evoked in the presence of either antagonist ruled out open channel block. These single-channel data confirm the hypothesis that both NMDA and glycine are coagonists of the NMDA receptor. Furthermore, the coagonist requirement increases the potential targets for therapeutic drugs aimed at blocking the pathologies resulting from overactivation of NMDA receptors.

Burst kinetics of single NMDA receptor currents in cell-attached patches from rat brain cortical neurons in culture.

1. The patch-clamp technique was used to record single-channel currents from cell-attached patches on rat brain cortical neurons in culture. The composition of the open and shut intervals during bursts of openings was studied in N-methyl-D-aspartate (NMDA) receptors exposed to 1 microM NMDA and 10 microM glycine at a membrane potential of -70 mV. 2. Open interval histograms were constructed for openings at each position (first, second, third, etc.) during all bursts. Distributions from openings two to five were fitted with similar (two or three) exponential components. The first opening from all bursts tended to be of shorter duration than the other openings. 3. Bursts were sorted according to the number of openings they contained and duration histograms were obtained for the openings at each position (one to five) during bursts of up to five openings. For bursts containing two or more openings, the distribution of open durations obtained at a given position were similar to each other regardless of the number of openings in the burst. 4. In bursts of two or more openings (up to five), duration histograms from the openings at each position in the burst were fitted with two or three exponential components that were similar for each opening. Bursts consisting of a single opening had a different distribution, having a relatively larger component of short duration. 5. Shut intervals during bursts were described by two exponential components with average time constants (and relative areas) (means +/- S.E.M.) of 0.06 +/- 0.01 ms (0.59 +/- 0.02) and 0.64 +/- 0.02 ms (0.41 +/- 0.02). Their distribution was independent of the numbers of openings in the bursts, their position within the burst, and the types of openings (long or short duration) contained within the burst. 6. These results suggest that each opening in bursts of two or more openings is kinetically similar to every other opening regardless of burst length. Analogously, each shut period during a burst was similar to every other. A kinetic model with three open and four closed intraburst states is shown to be consistent with these results for bursts of two or more openings.

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.

Single ion channel's view of classical receptor theory.

The nature of drug agonism has been the central mystery of two conceptually different approaches: classical receptor theory, which does not require any knowledge of mechanism, and mechanistic theories, which do. Ligand-activated ion channel macromolecules that contain both the agonist receptor site and molecular machinery to generate a response present a unique experimental system with which to explore agonism and antagonism. Electrical recordings from one channel at a time offer phenomena and a perspective quite different from that usually encountered in studies of the drug-receptor interaction. This review describes patch-clamp recordings that illustrate the ligand-evoked behavior that gives rise to classical phenomena. A comparison of the channel currents recorded in the presence of different agonists reveals how these drugs act as full or partial agonists. At higher concentrations of agonist, the conformational and kinetic transitions that underlie desensitization can be observed. Receptor conformational changes induced by agonist and antagonist binding further expand our ideas about what these drugs do, and contribute to the growth of concepts that will further our understanding of drug agonism.

Modulation of acetylcholine receptor desensitization by forskolin is independent of cAMP.

Biochemical and electrophysiological studies suggest that adenosine 3',5'-monophosphate (cAMP)-dependent phosphorylation of the nicotinic acetylcholine receptor channel is functionally significant because it modifies the receptor's rate of desensitization to acetylcholine. In studies that support this conclusion researchers have used forskolin to stimulate cAMP-dependent phosphorylation in intact muscle. It is now shown that although forskolin facilitated desensitization in voltage-clamped rat muscle, this effect was not correlated with the abilities of forskolin and forskolin analogs to activate adenylate cyclase or phosphorylate the receptor. Furthermore, elevation of intracellular cAMP or addition of the catalytic subunit of A-kinase failed to alter desensitization. Therefore, in intact skeletal muscle, cAMP-dependent phosphorylation does not modulate desensitization.

A comparison of calcium-activated potassium channel currents in cell-attached and excised patches.

Single channel currents from Ca-activated K channels were recorded from cell-attached patches, which were then excised from 1321N1 human astrocytoma cells. Cells were depolarized with K (110 mM) so that the membrane potential was known in both patch configurations, and the Ca ionophore A23187 or ionomycin (20-100 microM) was used to equilibrate intracellular and extracellular [Ca] (0.3 or 1 microM). Measurements of intracellular [Ca] with the fluorescent Ca indicator quin2 verified that [Ca] equilibration apparently occurred in our experiments. Under these conditions, where both membrane potential and intracellular [Ca] were known, we found that the dependence of the channel percent open time on membrane potential and [Ca] was similar in both the cell-attached and excised patch configuration for several minutes after excision. Current-voltage relations were also similar, and autocorrelation functions constructed from the single channel currents revealed no obvious change in channel gating upon patch excision. These findings suggest that the results of studies that use excised membrane patches can be extrapolated to the K-depolarized cell-attached configuration, and that the relation between [Ca] and channel activity can be used to obtain a quantitative measure of [Ca] near the membrane intracellular surface.

N-bromoacetamide removes a calcium-dependent component of channel opening from calcium-activated potassium channels in rat skeletal muscle.

Calcium-activated potassium channels from cultured rat skeletal muscle were treated with the protein-modifying reagent N-bromoacetamide (NBA) (0.3-1 mM) and studied in excised patches using patch-clamp techniques. After NBA treatment, channels opened only occasionally, and, in contrast to untreated channels, the open probability was no longer sensitive to intracellular surface calcium ions (1 nM to 100 microM). Channel activity did, however, exhibit a voltage dependence similar in direction and magnitude to that shown before NBA treatment (increasing e-fold with 19 mV depolarization). Distributions of open channel lifetimes revealed that NBA treatment virtually abolished openings of long duration, which suggests that this class of openings requires calcium sensitivity. These effects were not reversed by subsequent washing. Quantitatively similar open probability, voltage dependence, and open-interval distributions were observed in untreated channels in calcium-free medium. These results suggest that NBA removed a calcium-dependent component of channel opening, and that normal channels are able to open in the absence of significant intracellular calcium concentrations.

Calcium-activated potassium channels in rat muscle inactivate from a short-duration open state.

Single channel recording techniques were applied to the study of activation and inactivation of Ca2+-activated K+ channels in excised patches of membrane from rat muscle grown in culture. The concentration of intracellular surface Ca2+ was 0.6 microM in all experiments. The time course of the averaged open probability during depolarizing voltage steps of 1 s duration was biphasic for steps more positive than 20 mV; a rapid activation phase was followed by a much slower apparent inactivation with a single exponential time constant in the range of 400-800 ms. The peak open probability and degree of inactivation increased as the steps were made more positive (+30 to +80 mV) or the holding potential more negative (+30 to -40 mV). A conditional probability analysis of the open intervals immediately adjacent to the long-duration shut intervals resulting from inactivation revealed that transitions to and from the inactivated state occurred almost exclusively via a short-duration open state (mean lifetime less than 200 microseconds). The rate of transition from the short-duration open state to the inactivated state was rapid (typical rate constant 1879/s) and was sufficiently probable that as many as one out of every three short-duration openings were followed by inactivation. Normal (non-inactivating) closure of the channel from this open state was also rapid (rate constant 4003/s). At constant voltage (+50 mV) and Ca2+ (0.6 microM), the channel opened to the short-duration open state approximately every 75 ms, suggesting that the slow inactivation of the averaged open probability might have been limited in part by the rate at which the channel entered the short-duration open state.

Single channel recordings from calcium-activated potassium channels in cultured rat muscle.

Single channel recordings from cultured rat skeletal muscle have revealed a large conductance (230 pS) channel with a high selectivity for K+ over Na+. In excised patches of membrane, the probability of channel opening is sensitive to micromolar concentrations of calcium ions at the intracellular surface of the patch. Channel openings appear grouped together into bursts whose duration increases with Ca2+ and membrane depolarization. Statistical analysis of the individual open times during each burst showed that there are two distinct open states of similar conductance but dissimilar average lifetimes. These channels might contribute to a macroscopic calcium-activated potassium conductance in rat skeletal muscle and other preparations.

Burst kinetics of single calcium-activated potassium channels in cultured rat muscle.

Burst kinetics of single Ca-activated K channels in excised patches of surface membrane from cultured rat muscle were studied using the patch-clamp technique. Channel activity was separated into bursts using a calculated gap derived from the distribution of shut intervals. Shut intervals greater than the calculated gap were taken as gaps between bursts. The distribution of burst duration was described as the sum of two exponentials with mean durations of about 0.8 and 24 msec (1 microM-Cai, + 20 mV), suggesting two classes of bursts (short and long). The composition of short and long bursts was determined from comparisons of the distributions of open intervals, unit bursts (bursts of single openings), and openings/burst. Short bursts consisted mainly of single openings to the open channel state of short mean lifetime. Long bursts consisted of one or more openings to the (compound) open-channel state of long mean lifetime, plus, in fewer than 70% of the long bursts, one or more openings to the short open-channel state. The frequency of occurrence of bursts from each class first increased and then decreased with increasing [Ca]i, with the number of long bursts increasing at a greater rate than the number of short bursts. The number of openings/short burst was relatively independent of [Ca]i, while the number of openings/long burst increased, often more than linearly, with increasing [Ca]i. This increase arose almost entirely from an increase in openings to the long open state. These results suggest that openings to the long open state typically require the binding of three or more Ca ions, and openings to the short open state typically require the binding of at least one Ca ion. This is the case whether the openings occur in isolation as bursts of single openings or in bursts composed of both types of openings. An obvious burst of channel activity would occur when the channel opens and closes several times without losing all its bound Ca. The power relationship between [Ca]i and the percentage of time spent in the open state is accounted for in terms of the effects of [Ca]i upon mean channel open time, openings/burst, and burst rate. A model is presented that describes quantitatively many features of the burst kinetics of the Ca-activated K channel for constant [Ca]i.

Calcium dependence of open and shut interval distributions from calcium-activated potassium channels in cultured rat muscle.

The stochastic properties of single Ca-activated K channels in excised patches of surface membrane from cultured rat muscle cells were studied using the patch-clamp technique. The distribution of all open intervals was described by the sum of two exponential distributions of short and long mean open time, suggesting at least two major open-channel states. Increasing the concentration of Ca at the inner membrane surface, [Ca]i, increased the mean duration of the long open distribution, while having little effect on the mean duration of the short open distribution. The frequency of openings to each distribution increased with [Ca]i. The rate of increase was a much steeper function of [Ca]i for openings in the long open distribution than for openings in the short open distribution; about 80% of the openings were to the long open distribution with 0.1 microM-Cai, increasing to 97% with 1 microM-Cai (+ 30 mV). These results suggest that openings in both open distributions are Ca-dependent, with openings in the long open distribution requiring the binding of more Ca ions than openings in the short open distribution. The distribution of all shut intervals at 0.5 microM-Cai and + 30 mV was described by the sum of three exponential distributions with mean durations of: 0.21 msec (short shut distribution), 1.90 msec (intermediate shut), and 44 msec (long shut). These results indicate that the channel typically enters at least three closed channel states during normal channel activity. In addition, a few longer shut intervals not accounted for by the above distributions suggested that there was a fourth infrequently occurring inactivated closed-channel state. The mean duration of the distribution of long shut intervals decreased with a power of about 2 with increasing [Ca]i under conditions where most openings were to the long open state (+ 30 mV, 0.25-1 microM-Cai). This observation suggests that openings to the long open distribution typically require the binding of two or more Ca ions. The mean intermediate shut interval appeared to increase slightly with increasing [Ca]i while the mean short shut interval was relatively Cai-independent. The percentage of all shut intervals that were short shut intervals increased with increasing [Ca]i while the percentage of long shut intervals decreased.(ABSTRACT TRUNCATED AT 400 WORDS)

Properties of single calcium-activated potassium channels in cultured rat muscle.

1. Properties of the Ca-activated K channel were studied in excised patches of surface membrane from cultured rat muscle cells using single channel recording techniques.2. Increasing the concentration of calcium at the intracellular membrane surface [Ca](i), increased both the frequency and effective duration of channel openings. Ca at the extracellular membrane surface was not sufficient to activate the channels.3. An approximate third power relationship (slope = 2.7) was observed between [Ca](i) and the percentage of time the channels spent in the open state.4. Both the frequency and effective duration of channel openings increased as the intracellular membrane surface was made more positive; the percentage of time spent in the open state increased e-fold for a 15 mV depolarization for low levels of activity.5. The percentage of time spent with 1, 2,...n channels open in membrane patches with n channels was described by the binomial distribution, suggesting that the channels opened and shut independently of one another.6. Single channel conductance (144 mM-K on both sides of the membrane) was essentially independent of membrane potential (-50 to +50 mV) and [Ca](i) (0.1 muM -1 mM), but did increase with temperature, from 100 pS at 1 degrees C to 300 pS at 37 degrees C.7. Channel activity occurred in apparent bursts, with the duration of the apparent bursts increasing with increasing [Ca](i).8. Two exponentials were required to describe the distribution of observed channel open times, suggesting two different open channel states of apparently normal conductance. The observed mean channel open time of these states at +30 mV was 0.34 and 2.2 msec with 0.1 muM-Ca(i) and was 0.47 and 6.9 msec with 0.5 muM-Ca(i).9. The channel occasionally entered an apparent third open channel state with a single channel current amplitude about 40% the amplitude of the normally observed single channel currents. The reduced conductance state was immediately preceded and followed by a normal conducting state.10. While the kinetics of the Ca-activated K channel appear complex, its large conductance and high Ca and voltage sensitivity suggest that it is uniquely suited to resist depolarizations of the cell membrane potential that are accompanied by increases in intracellular Ca.

A study of desensitization of acetylcholine receptors using nerve-released transmitter in the frog.

1. Desensitization of acetylcholine (ACh) receptors was studied at the frog neuromuscular junction under voltage clamp.2. ACh was applied directly to junctional receptors by stimulating the motor nerve with trains of impulses. End-plate currents (e.p.c.s) were used to estimate the total number of channel openings by the junctional ACh receptors, and miniature end-plate currents (m.e.p.c.s) were used to measure changes in post-synaptic sensitivity. Under the conditions of these experiments the changes in m.e.p.c. amplitudes were shown to be post-synaptic in origin and thus provided a measure of desensitization.3. When the acetylcholinesterase was inhibited with diisopropylfluorophosphate, neostigmine, or collagenase treatment to prolong the duration of the nerve-released ACh in the synaptic cleft, desensitization developed during repetitive stimulation of 1000 impulses at 5-33 impulses/sec and then recovered after the conditioning trains, with a time constant of about 25 sec.4. When the acetylcholinesterase was active so that the duration of ACh in the synaptic cleft resulting from each nerve impulse was brief (< 300 musec), desensitization developed in response to 300-500 pairs of nerve stimuli if the interval between the impulses of each pair was 25 msec or less. When the interval was 30 msec or greater, however, measurable desensitization did not occur, even if the total number of channel openings was many times greater than in the experiments with shorter intervals or inhibited esterase where desensitization readily occurred.5. The desensitization observed to pairs of impulses was enhanced by chlorpromazine and decreased when the post-synaptic membrane was depolarized, properties similar to those described previously for desensitization to bath and ionophoretic application of ACh.6. These results indicate that desensitization to nerve-released transmitter is not a simple consequence of receptor activation, is not due to blockade of the open receptor channels by ACh, and does not result from ACh binding directly to desensitized receptors with a resulting shift in the receptor population towards the desensitized state.7. We suggest that the desensitization observed to nerve-released transmitter is a two-step process with both steps initiated by ACh. In the first step ACh converts some receptors into a desensitizable state which has an apparent lifetime of less than 30 msec; in the second step ACh desensitizes the desensitizable state.

The effect of (+)-tubocurarine on neuromuscular transmission during repetitive stimulation in the rat, mouse, and frog.

1. The effect of tubocurarine on amplitudes of end-plate currents in response to trains of repetitive stimulation (50-150/sec) was investigated in voltage-clamped muscle fibres of the rat, mouse and frog. 2. In rat and mouse muscle, the presence of tubocurarine led to a more rapid decline (rundown) in the amplitudes of successive end-plate currents during trains of impulses. In frog, tubocurarine caused an increase in apparent facilitation of end-plate current amplitudes during the first few impulses of repetitive stimulation; this increase was followed by a more rapid rundown of end-plate current amplitude. 3. These effects of tubocurarine appear not to be an artifact resulting from inadequate control of membrane potential in voltage-clamped fibres. 4. The more rapid rundown during trains of end-plate currents in the presence of tubocurarine showed little variation with membrane potential indicating that voltage-sensitive channel blockade by tubocurarine was not a major factor contributing to the rundown. 5. The effect of tubocurarine on the apparent facilitation and rundown of end-plate current amplitudes was typically decreased by reducing the frequency of stimulation. 6. These results suggest that tubocurarine affects transmitter release at neuromuscular junctions during repetitive stimulation.

The effects of external Ca++ and Mg++ on the voltage sensitivity of desensitization in Electrophorus electroplaques.

Desensitization onset was studied in voltage-clamped Electrophorus electroplaques during prolonged exposure to bath-applied carbamylcholine. The time-course of desensitization was described by a first-order rate constant kappa obs, which increased exponentially with membrane hyperpolarization from -20 to -90 mV. When Ca++ was increased from 2 to 10 mM, the voltage sensitivity of kappa obs decreased; kappa obs decreased for voltages more negative than -40 mV, and increased slightly at voltages more positive than -40 mV. 10 mM Mg++ had a less pronounced effect and the voltage sensitivity of kappa obs was unchanged. The equilibrium level of desensitization, estimated from the carbamylcholine-dependent conductance which remained after desensitization was apparently complete, also increased with hyperpolarization.