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D Colquhoun

Publications and source records attributed to D Colquhoun.

At least 37 records · Page 2Linked to original sources
PubMed1996

Single-channel currents from recombinant NMDA NR1a/NR2D receptors expressed in Xenopus oocytes.

We have investigated the single-channel and whole-cell behaviour of recombinant N-methyl-D-aspartate (NMDA) receptors formed from NR1a and NR2D receptor subunits expressed in Xenopus oocytes. The EC50 for apparent steady-state activation of NR1a/NR2D receptors by glutamate was 450 nM, while extracellular MG2+ produced a voltage-dependent block of glutamate responses with an IC50 of 440 microM at -70 mV. At negative holding potentials glutamate-activated NR1a/NR2D single-channel currents, in 0.85 mM external Ca2+, had slope conductances of 35 pS for the main level, and 17 pS for the sublevel; direct transitions occurred between these two conductance levels. On average 35 pS events had mean open times of 1.01 +/- 0.04 ms, whereas the mean open times of 17 pS events were consistently longer (1.28 +/- 0.06 ms). In 5 mM external Ca2+ the larger conductance level was reduced to 20 pS whereas in Ca(2+)-free solutions it was increased to 50 pS. The frequency of transitions between the main and subconductance levels showed temporal asymmetry: 35-17 pS transitions were more frequent (61%) than 17-35 pS transitions. This asymmetry was not affected by alterations in the external Ca2+ concentration (up to 5 mM). In conclusion, the NR1a/NR2D channel is, like NR1a/NR2C, a 'low conductance' NMDA channel, but it can be distinguished from NR1a/NR2C channels on the basis of transition asymmetry and differences in the open times of its main and sub-conductance levels.

Animals↗
PubMed1996

Deactivation and desensitization of non-NMDA receptors in patches and the time course of EPSCs in rat cerebellar granule cells.

1. Spontaneous and evoked non-NMDA receptor-mediated EPSCs were recorded from cerebellar granule cells in slices at approximately 24 and approximately 34 degrees C. The EPSC decay was fitted with the sum of two exponential functions. 2. The time courses of non-NMDA receptor deactivation and desensitization were determined with fast concentration jumps of glutamate onto patches from cultured granule cells. Deactivation (decay time constant tau = 0.6 ms at 24 degrees C) was substantially faster than desensitization (tau = 4 ms). Both processes were fitted by single exponential functions. 3. The decay of the fast component of the spontaneous EPSC (tau EPSCfast = 0.9 ms at 23 degrees C) was marginally slower than deactivation but too fast to be determined by desensitization. Our results suggest that the decay of this component is set by both the rate of decline of transmitter concentration and channel deactivation. 4. A simple diffusion model predicts that the time course of transmitter in the cleft declines slowly during the later stages of its action. The slow phase of transmitter removal could account for the time course of the slow component of the spontaneous EPSC (tau EPSCslow = 8 ms at 23 degrees C).

Animals↗
PubMed1995

Persistence: a new statistic for characterizing ion-channel activity.

We introduce and illustrate by examples a new statistical technique, the persistence function, for characterizing ion-channel activity in a single-channel patch-clamp recording. Persistence is a function of both current and time. It is the probability that the current is at a given level (conditional on it having been at that level at an earlier time). Viewed as a function of current it exhibits the prominent conductance levels present in the recording, and viewed as a function of time for a conductance level it portrays the kinetics at that level.

Ion Channels↗
PubMed1995

Determination of NMDA NR1 subunit copy number in recombinant NMDA receptors.

Co-expression of wild-type and mutated NMDA NR1 (N598R) subunits in Xenopus oocytes has been used to determine the stoichiometry of the NMDA receptor-channel. When expressed together, wild-type NR2A and mutant NR1 (N598R) subunits produced channels with a main conductance of 2.6 pS and a sublevel of 1.2 pS. These conductances were clearly different from those obtained from wild-type NR1 and wild-type NR2A channels which gave characteristic 50 pS events with a 40 pS sublevel. When wild-type and mutant NR1 subunits were co-expressed together with NR2A subunits a different channel type with a main conductance of 15.2 pS and a sublevel of 11.4 pS was obtained, as well as the 'all wild-type' and 'all mutant' channels described above. These results indicate that there are likely to be two copies of the NR1 subunit in each NMDA receptor complex.

Animals↗
PubMed1995

Desensitization of N-methyl-D-aspartate receptors: a problem of interpretation.

The phenomenon of desensitization is universal, but its mechanism is still ill-understood and controversial. A recently published study [Lin, F. & Stevens, C. F. (1994) J. Neurosci, 14, 2153-2160] attempted to cast light on the mechanism of desensitization of N-methyl-D-aspartate (NMDA) receptors, in particular the vexed question of whether the channel must open before it can desensitize. During the desensitizing preexposure to agonist in those experiments, more desensitization was produced when channel openings were observed than when no openings were observed. The conclusion that "desensitization occurs more rapidly from the open state" unfortunately was based on a stochastic fallacy, and we present here a theoretical treatment and illustration showing that the observed behavior is predicted by a simple mechanism in which desensitization can occur only from a shut state.

Animals↗
PubMed1995

A P2X purinoceptor expressed by a subset of sensory neurons.

ATP is known to depolarize sensory neurons, and may play a role in nociceptor activation when released from damaged tissue. Here we report the molecular cloning and characterization of a new member of the P2X receptor family, P2X3, expressed by these cells. The channel transcript was present in a subset of rat dorsal-root-ganglion sensory neurons, some of which express nociceptor-associated markers; it was absent in other tissues that were tested, including sympathetic, enteric and central nervous system neurons. Moreover, when expressed in Xenopus oocytes, the channel showed an ATP-dependent cation flux. P2X3 is the only ligand-gated channel known to be expressed exclusively by a subset of sensory neurons. The remarkable selectivity of expression of the channel coupled with its sensory neuron-like pharmacology suggests that this channel may transduce ATP-evoked nociceptor activation.

Amino Acid Sequence↗
PubMed1995

Heterogeneity of neuronal nicotinic acetylcholine receptors in thin slices of rat medial habenula.

1. Neuronal nicotinic acetylcholine receptors in slices of rat medial habenula were studied using patch clamp recording techniques. 2. Whole cell current responses to cytisine could be blocked by hexamethonium, as expected for nicotinic receptors. The whole cell current-voltage relations were linear at negative membrane potentials, but showed strong inward rectification when chloride currents were minimized. 3. When 1 mM Ca2+ (0 mM Mg2+) was present in the external recording solution, the single channel conductances elicited by acetylcholine or nicotine in twenty patches were in the range 39-58 pS, with a mean of 47 pS. There appeared to be at least two groups of conductances. 4. In the open point amplitude distributions of three patches, the most common amplitude corresponded to 41 pS (81% of the area). In another four patches the most common amplitude corresponded to a mean conductance of 51 pS (83% of the area). Direct transitions between open levels were rare. 5. Channel closed times were not significantly different for the two conductance groups. However, for the four patches with predominantly 51 pS openings, the means of the distributions of open times longer than two filter rise times averaged 5.8 ms. Those patches with predominantly 41 pS openings averaged 14 ms. Also, for patches with predominantly 51 pS openings the overall mean burst length was 5.8 ms, whereas for patches with predominantly 41 pS openings it was 16.1 ms. 6. These observations suggest that 51 and 41 pS openings result from the activity of at least two, but possibly more, different receptor subtypes. We conclude that nicotinic receptors in the rat ventral medial habenula are heterogeneous.

Acetylcholine↗
PubMed1994

Comparison of neuronal nicotinic receptors in rat sympathetic neurones with subunit pairs expressed in Xenopus oocytes.

1. The agonist sensitivity of nicotinic acetylcholine receptors in rat superior cervical ganglion (SCG) neurones was compared with that of cloned receptors expressed in Xenopus oocytes by pairwise injections of alpha 3-beta 2 or alpha 3-beta 4 neuronal nicotinic subunit combinations. 2. Agonist responses in rat SCG neurones indicated that cytisine was the most potent agonist and lobeline the least potent (rank order of potency: cytisine > dimethylphenylpiperazinium iodide (DMPP) > nicotine > ACh > carbachol > lobeline). 3. Receptors expressed in oocytes by injection of alpha 3 and beta 2 subunits had a relatively high sensitivity to DMPP and low sensitivity to cytisine (rank order of potency: DMPP > ACh > lobeline > carbachol > nicotine > cytisine), whereas receptors composed of alpha 3 and beta 4 subunits had a high sensitivity to cytisine and low sensitivity to DMPP (rank-order of potency: cytisine > nicotine approximately ACh > DMPP > carbachol > lobeline). 4. With the exception of responses to DMPP, agonist sensitivity measurements suggest that nicotinic receptors in the rat SCG are composed of alpha 3 and beta 4 subunits. The results are discussed in terms of the receptor subunit mRNAs known to be expressed in the rat SCG and previous evidence of functional heterogeneity of rat SCG nicotinic acetylcholine receptors.

Alkaloids↗
PubMed1994

Single channel properties of cloned NMDA receptors in a human cell line: comparison with results from Xenopus oocytes.

1. Human embryonic kidney (HEK) 293 cells were transiently transfected with cDNAs encoding the NR1a-NMDA epsilon 1[NR2A] subunit combination of the NMDA receptor. Single channel behaviour was recorded from outside-out membrane patches, with the aim of comparing the results with those, recorded under the same conditions, from Xenopus oocytes injected with messenger RNA coding for the NR1a-NR2A combination. 2. Single channels in HEK 293 cells showed a main conductance level of 51.4 +/- 2.4 pS, compared with 50.1 +/- 1.4 pS for channels in oocytes. A subconductance level of 38.1 +/- 2.1 pS was found in HEK 293 cells, compared with 38.3 +/- 1.3 pS in oocytes. The frequencies of transitions between the shut and the two conductance levels were also very similar. 3. Distributions of shut times could be fitted with five exponential components. In HEK 293 cells the first three of these components had time constants of 39 +/- 4 microseconds, 0.54 +/- 0.04 ms and 9.94 +/- 1.3 ms; in oocytes the values were 69 +/- 35 microseconds, 0.54 +/- 0.15 ms and 6.53 +/- 4.6 ms, respectively. The relative areas of the components were also similar in the two systems. 4. The distribution of all apparent open times for the sublevels was fitted with two exponential components giving time constants of 0.18 +/- 0.02 ms and 1.31 +/- 0.17 ms (for HEK cells) or of 0.31 +/- 0.36 ms and 1.31 +/- 1.1 ms (for oocytes).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Hypertension and heart disease. The need for clear thinking.

Hypertension is a major risk factor for vascular disease-cerebral, cardiac and peripheral. The systolic blood pressure is the most important prognostic factor. An extensive work-up searching for a cause is not indicated. Controlling hypertension has been shown to decrease incidence of stroke, heart failure, myocardial infarction and sudden death. Thiazide and beta-blockers have stood the test of time and have the best track record in preventing complications of hypertension. Surrogate endpoints of therapy, such as effect on insulin resistance, are interesting from an academic point of view. But they are no substitute for randomised clinical trials and the real endpoints of stroke, myocardial infarction and sudden death.

Adrenergic beta-Antagonists↗
PubMed1992

Single-channel conductances of NMDA receptors expressed from cloned cDNAs: comparison with native receptors.

To cast light on the subunit composition of native NMDA-type glutamate receptors, four cloned subunits of the NMDA receptor have been expressed, in pairs, in Xenopus oocytes, and their single-channel properties have been measured. The conductances of the channels, and their characteristic patterns of sublevel transitions, turn out to be useful diagnostic criteria for subunit composition. The NR1-NR2A and NR1-NR2B combinations (which have identical TM2 sequences) are very similar to each other. Both have 50 pS openings and brief 40 pS sublevels (in 1 mM external Ca2+), with similar mean lifetimes and frequencies. They also show close quantitative resemblance to the channels of hippocampal CA1 and dentate gyrus cells and of cerebellar granule cells, except that the NR1-NR2A combination has a lower glycine sensitivity than the native channels. In contrast, the NR1-NR2C combination produces a channel with 36 pS and 19 pS conductances of similar (brief) duration; these closely resemble the 38-18 pS channels that have previously been observed in large cerebellar neurons in culture (together with 50 pS channels).

Animals↗
PubMed1992

Rapid decay of averaged single-channel NMDA receptor activations recorded at low agonist concentration.

The NMDA class of glutamate receptors have the unique property of binding some agonists, including glutamate, for a very long period of time. One manifestation of this is that brief (1 ms) application of glutamate (1 mM) produces a slowly decaying current, the major component of which has a time constant of approximately 200 ms. Application of glutamate at low concentrations allows identification of groups ('superclusters') of openings in the data record that probably correspond to a single period during which one or more molecules of glutamate are bound to the receptor, i.e. a single activation of the channel. The length of such superclusters is long on average (74 ms); the longest component of the distribution has a duration of approximately 300 ms, and comprises about 25% of the area. However, aligning many superclusters to obtain an average current reveals that the decay is mainly fast; the major component has a time constant of around only 5 ms. It is shown that incorporation of a distribution of first latencies (from the time of the jump to the first opening) can explain at least part of this discrepancy.

Animals↗