Stochastic Properties of Ion Channel Openings and Bursts in a Membrane Patch that Contains Two Channels: Evidence Concerning the Number of Channels Present when a Record Containing Only Single Openings is Observed
If a single ion channel record is observed in which two ion channels are never simultaneously open, then it is often of interest to know whether the observations indeed arose from the activity of only one ion channel. This question can be answered if it is possible to calculate the distribution of the duration of runs of single openings in a membrane patch that contains two active channels. If the observed run of single openings is much longer than that expected for a patch with two channels it is likely that only one channel was active. An approximate method is presented for calculating the distribution of the duration of runs of single openings in a patch with two active channels; this method has the advantage that it can be calculated from observable quantities, and requires no knowledge of the details of the ion-channel mechanism or its rate constants. The accuracy of this approximation is tested by exact calculations of the properties of runs of single openings, and of single bursts, for two specific mechanisms and a large range of rate constants. The approximation is good in all cases in which openings occur singly, or in closely spaced bursts. If, as is common in practice, openings occur in clusters that are separated by long shut periods, then overlap of clusters from two different channels may be detected, if no double opening is produced, as a period in the middle of a cluster in which the probability of being open doubles. The results derived here can be applied to such a period to test whether it results from the simultaneous activity of two channels, rather than from a change in the properties of a single channel.
Available from: Kasper B Hansen
- "For analysis, the recordings were prefiltered at 8 kHz (−3 dB), digitized at 40 kHz, and analyzed using the time course fitting method (SCAN software provided by Dr. David Colquhoun, University College London, http://www.ucl.ac.uk/Pharmacology/dcpr95.html). Open and closed duration histograms were constructed with an imposed resolution for open and shut durations of 53 μs and 31 μs, respectively (Colquhoun and Hawkes, 1990). The amplitude distributions were fitted to the sum of multiple Gaussian components and apparent open and closed duration distributions fitted to the sum of multiple exponential components using the maximum likelihood (EKDIST, http://www.ucl.ac.uk/Pharmacology/dcpr95.html; ChanneLab, Synaptosoft). "
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ABSTRACT: NMDA receptors comprised of different NR2 subunits exhibit strikingly unique biophysical and pharmacological properties. Here, we report that the extracellular amino-terminal domain (ATD) of the NR2 subunit controls pharmacological and kinetic properties of recombinant NMDA receptors, such as agonist potency, deactivation time course, open probability (P(OPEN)), and mean open/shut duration. Using ATD deletion mutants of NR2A, NR2B, NR2C, NR2D, and chimeras of NR2A and NR2D with interchanged ATD [NR2A-(2D-ATD) and NR2D-(2A-ATD)], we show that the ATD contributes to the low glutamate potency of NR2A-containing NMDA receptors and the high glutamate potency of NR2D-containing receptors. The ATD influences the deactivation time courses of NMDA receptors, as removal of the ATD from NR2A slows the deactivation rate, while removal of the ATD from NR2B, NR2C and NR2D accelerates the deactivation rate. Open probability also is influenced by the ATD. Removal of the ATD from NR2A or replacement of the NR2A-ATD with that of NR2D decreases P(OPEN) in single-channel recordings from outside-out patches of HEK 293 cells. In contrast, deletion of the ATD from NR2D or replacement of the NR2D ATD with that of NR2A increases P(OPEN) and mean open duration. These data demonstrate the modular nature of NMDA receptors, and show that the ATD of the different NR2 subunits plays an important role in fine-tuning the functional properties of the individual NMDA receptor subtypes.
Available from: Samantha Pitt
- "Because it is impossible to estimate the number of channels in the patch for our sort of channel, the only data used for global mechanism fits were sequences of events that were likely to reflect the activity of one channel molecule. At high glycine concentrations (50 μM and 1 mM), this means selecting stretches of high Popen data (i.e., desensitization clusters): these have to come from a single channel because the Popen at these concentration is so high that we would have to see doubles if the activity came from more than one channel (Colquhoun and Hawkes, 1990). At the lower concentration (1 μM), openings occur in bursts. "
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ABSTRACT: The time course of currents mediated by native and recombinant glycine receptors was examined with a combination of rapid agonist applications to outside-out patches and single-channel recording. The deactivation time constant of currents evoked by brief, saturating pulses of glycine is profoundly affected by the chloride concentration on the intracellular side of the cell membrane. Deactivation was threefold slower when intracellular chloride was increased from a low level (10 mm), similar to that observed in living mature neurons, to 131 mm ("symmetrical" chloride, often used in pipette internal solutions). Single-channel analysis revealed that high chloride has its greatest effect on the channel closing rate, slowing it by a factor of 2 compared with the value we estimated in the cell-attached mode (in which the channels are at physiological intracellular chloride concentrations). The same effect of chloride was observed when glycinergic evoked synaptic currents were recorded from juvenile rat spinal motoneurons in vitro, because the decay time constant was reduced from approximately 7 ms to approximately 3 ms when cells were dialyzed with 10 mm chloride intracellular recording solution. Our results indicate that the time course of glycinergic synaptic inhibition in intact neurons is much faster than is estimated by measurements in symmetrical chloride and can be modulated by changes in intracellular chloride concentration in the range that can occur in physiological or pathological conditions.
Available from: ncbi.nlm.nih.gov
- ", continuous line) accurately predicts the behaviour of channels with NR1/NR2C-like properties, and thus can be used as an analytical tool to assess probability that patches contain a single active channel. The three dashed lines indicate the upper limit for P = 0.0044, 0.0132 and 0.0392 calculated as 3.24E r , 4.33E r and 5.43E r , respectively (Colquhoun & Hawkes, 1990). We analysed experimental data from two patches in which we observed simultaneous double openings but never simultaneous triple openings. "
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ABSTRACT: The N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptors comprises both NR1 and NR2 subunits, and plays numerous roles in both physiological and pathophysiological processes in the central nervous system (CNS). NR2C-containing NMDA receptors are most abundant in cerebellum, thalamus and olfactory bulb, and are also expressed in oligodendrocytes and hippocampal interneurons. We have used patch clamp recording to explore the activation properties of recombinant NR1/NR2C receptors expressed in HEK293 cells. NR1/NR2C receptors activated by a maximally effective concentration of glutamate and glycine had two main conductance levels of 45 pS and 28 pS when the extracellular Ca(2+) concentration was 0.5 mm and the holding potential was -80 mV. The occurrence of the lower subconductance state was reduced in the absence of extracellular Ca(2+). The distribution of closed durations recorded from patches with a high probability of containing only one active channel were best fitted by five exponential functions; the apparent open duration histogram could be fitted by two exponential functions (n = 10 patches). The apparent mean open time of NR1/NR2C receptors was brief (0.52 +/- 0.04 ms), suggesting that the stability of the open state of the NR1/NR2C receptors is lower than other NR2-containing receptors. NR1/NR2C open probability was exceptionally low, being 0.011 +/- 0.002 in patches containing a single active receptor (n = 8). Fast agonist concentration jumps were performed on outside out patches with multiple NR1/NR2C channels, which activated with a 10-90% rise time of 3.9 +/- 0.4 ms, faster than other NR2-containing receptors. The deactivation time constant after a brief (5-8 ms) application of a maximally effective concentration of agonists was 319 +/- 34 ms. The majority of the patches also showed a modest level of desensitization that could be described by either a single or a double exponential time course with the fastest time constant between 15 and 47 ms. Conceptual models of activation were fitted using the maximum interval likelihood (MIL) method to the sequence of open and closed durations recorded from outside-out patches that contained one active NR1/NR2C channel. NR1/NR2C receptor properties including modest desensitization and low open probability could be described by gating schemes similar to those previously proposed for other NMDA receptor subunit combinations.
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