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D Colquhoun

Publications and source records attributed to D Colquhoun.

At least 19 recordsLinked to original sources
PubMed2003

The quality of maximum likelihood estimates of ion channel rate constants.

Properties of maximum likelihood estimators of rate constants for channel mechanisms are investigated, to see what can and cannot be inferred from experimental results. The implementation of the HJCFIT method is described; it maximises the likelihood of an entire sequence of apparent open and shut times, with the rate constants in a specified reaction mechanism as free parameters. The exact method for missed brief events is used. Several methods for testing the quality of the fit are described. The distributions of rate constants, and correlations between them, are investigated by doing sets of 1000 fits to simulated experiments. In a standard nicotinic receptor mechanism, all nine free rate constants can be estimated even from one single channel recording, as long as the two binding sites are independent, even when the number of channels in the patch is not known. The estimates of rate constants that apply to diliganded channels are robust; good estimates can be obtained even with erroneous assumptions (e.g. about the value of a fixed rate constant or the independence of sites). Rate constants that require distinction between the two sites are less robust, and require that an EC50 be specified, or that records at two concentrations be fitted simultaneously. Despite the complexity of the problem, it appears that there exist two solutions with very similar likelihoods, as in the simplest case. The hazards that result from this, and from the strong positive correlation between estimates of opening and shutting rates, are discussed.

Binding Sites↗
PubMed2003

Properties of the human muscle nicotinic receptor, and of the slow-channel myasthenic syndrome mutant epsilonL221F, inferred from maximum likelihood fits.

The mechanisms that underlie activation of nicotinic receptors are investigated using human recombinant receptors, both wild type and receptors that contain the slow channel myasthenic syndrome mutation, epsilonL221F. The method uses the program HJCFIT, which fits the rate constants in a specified mechanism directly to a sequence of observed open and shut times by maximising the likelihood of the sequence with exact correction for missed events. A mechanism with two different binding sites was used. The rate constants that apply to the diliganded receptor (opening, shutting and total dissociation rates) were estimated robustly, being insensitive to the exact assumptions made during fitting, as expected from simulation studies. They are sufficient to predict the main physiological properties of the receptors. The epsilonL221F mutation causes an approximately 4-fold reduction in dissociation rate from diliganded receptors, and a smaller increase in opening rate and mean open time. These are sufficient to explain the approximately 6-fold slowing of decay of miniature synaptic currents seen in patients. The distinction between the two binding sites was less robust, the estimates of rate constants being dependent to some extent on assumptions, e.g. whether an extra short-lived shut state was included or whether the EC50 was constrained. The results suggest that the two binding sites differ by roughly 10-fold in the affinity of the shut receptor for ACh in the wild type, and that in the epsilonL221F mutation the lower affinity is increased so the sites become more similar.

Acetylcholine↗
PubMed2002

Recessive inheritance and variable penetrance of slow-channel congenital myasthenic syndromes.

BACKGROUND: Slow-channel congenital myasthenic syndromes (SCCMS) typically show dominant inheritance. They are caused by missense mutations within the subunits of muscle nicotinic acetylcholine receptors (AChR) that result in prolonged ion channel activations. SCCMS mutations within the AChR subunit are located in various functional domains, whereas fully described mutations in AChR non- subunits have, thus far, been located only in the M2 channel-lining domain. The authors identified and characterized two -subunit mutations, located outside M2, that underlie SCCMS in three kinships. In two of the three kinships, the syndrome showed an atypical inheritance pattern. METHODS: These methods included clinical diagnosis, mutation detection, haplotype analysis, and functional expression studies using single-channel recordings of mutant AChR transiently transfected into HEK293 cells. RESULTS: The authors identified two SCCMS mutations in the AChR subunit, L78P and L221F. Both mutations prolonged ACh-induced ion channel activations. L78P is present in a consanguineous family and appears to be pathogenic only when present on both alleles, and L221F shows variable penetrance in one of the two families that were identified harboring this mutation. CONCLUSION: SCCMS mutations may show a recessive inheritance pattern and variable penetrance. A diagnosis of SCCMS should not be ruled out in cases of CMS with an apparent recessive inheritance pattern.

Adolescent↗
PubMed2001

Long-term risk stratification for survivors of acute coronary syndromes. Results from the Long-term Intervention with Pravastatin in Ischemic Disease (LIPID) Study. LIPID Study Investigators.

OBJECTIVES: We developed a prognostic strategy for quantifying the long-term risk of coronary heart disease (CHD) events in survivors of acute coronary syndromes (ACS). BACKGROUND: Strategies for quantifying long-term risk of CHD events have generally been confined to primary prevention settings. The Long-term Intervention with Pravastatin in Ischemic Disease (LIPID) study, which demonstrated that pravastatin reduces CHD events in ACS survivors with a broad range of cholesterol levels, enabled assessment of long-term prognosis in a secondary prevention setting. METHODS: Based on outcomes in 8,557 patients in the LIPID study, a multivariate risk factor model was developed for prediction of CHD death or nonfatal myocardial infarction. Prognostic indexes were developed based on the model, and low-, medium-, high- and very high-risk groups were defined by categorizing the prognostic indexes. RESULTS: In addition to pravastatin treatment, the independently significant risk factors included: total and high density lipoprotein cholesterol, age, gender, smoking status, qualifying ACS, prior coronary revascularization, diabetes mellitus, hypertension and prior stroke. Pravastatin reduced coronary event rates in each risk level, and the relative risk reduction did not vary significantly between risk levels. The predicted five-year coronary event rates ranged from 5% to 19% for those assigned pravastatin and from 6.4% to 23.6% for those assigned placebo. CONCLUSIONS: Long-term prognosis of ACS survivors varied substantially according to conventional risk factor profile. Pravastatin reduced coronary risk within all risk levels; however, absolute risk remained high in treated patients with unfavorable profiles. Our risk stratification strategy enables identification of ACS survivors who remain at very high risk despite statin therapy.

Aged↗
PubMed2000

Effects of pravastatin in 3260 patients with unstable angina: results from the LIPID study.

BACKGROUND: The LIPID study is a major trial of secondary prevention of coronary-heart-disease events that includes hospital admission with unstable angina (as well as myocardial infarction) as a qualifying event. In this substudy of LIPID, we compared subsequent cardiovascular risks and the effects of pravastatin in patients with previous unstable angina or previous myocardial infarction. METHODS: 3260 patients diagnosed with unstable angina and 5754 with acute myocardial infarction 3-36 months previously were randomly assigned 40 mg pravastatin daily or placebo over a mean of 6.0 years. The risk reduction of a range of cardiovascular events was estimated by means of the hazard ratio in Cox's proportional hazards model. FINDINGS: Among patients assigned placebo, survival in the two diagnosis groups was similar. The relative risk reduction for mortality with pravastatin was 20.6% in the myocardial infarction group and 26.3% in the unstable angina group (p=0.55). Pravastatin significantly reduced the rates of all prespecified coronary endpoints in the myocardial infarction group. In patients with previous unstable angina, coronary heart disease mortality, total mortality, myocardial infarction, a need for coronary revascularisation, the number of admissions to hospital, and the number of days in hospital were significantly lower with pravastatin. Overall, hospital admission for unstable angina was the most common endpoint (24.6% of the placebo group; 22.3% of the pravastatin group). INTERPRETATION: Patients who have survived acute myocardial infarction or unstable angina have a similar long-term prognosis, a high occurrence of subsequent unstable angina, and benefit similarly from therapy with pravastatin.

Adult↗
PubMed2000

Single-channel analysis of an NMDA receptor possessing a mutation in the region of the glutamate binding site.

Recombinant NR1a/NR2A(T671A) N-methyl-D-aspartate (NMDA) receptor-channels, which carry a point mutation in the putative glutamate binding site that reduces glutamate potency by around 1000-fold, have been expressed in Xenopus laevis oocytes and their single-channel properties examined using patch-clamp recording techniques. Shut time distributions of channel activity were fitted with a mixture of five exponential components. The first three components in each distribution were considered to occur within a channel activation as they exhibited little or no dependence on agonist concentration. Bursts of single-channel openings were defined by a critical gap length with a mean of 5.65 +/- 0.37 ms. Shut intervals with a duration longer than this value were considered to occur between separate bursts of channel openings. Distributions of the lengths of bursts of openings were fitted with a mixture of four exponential components. The longest two components carried the majority of the charge transfer in the channel recordings and had means of 7.71 +/- 1.1 and 37.7 +/- 4.3 ms. The overall probability of a channel being open during a burst was high (mean 0.92 +/- 0.01). Brief concentration jumps (1 ms) of 10 mM glutamate were applied to outside-out patches so that a comparison between the macroscopic current relaxation and steady-state single-channel activity evoked by glutamate could be made. The decay of such macroscopic currents was fitted with a single exponential component with a mean of 32.0 +/- 3.53 ms. The good agreement between macroscopic current decay following brief agonist exposure and the value for the slowest component of the burst length distribution suggests that the bursts of openings that we identified in steady-state recordings represent individual activations of recombinant NR1a/NR2A(T671A) NMDA receptor-channels. A new way of displaying geometric distributions is suggested, and the utility of a modified definition of the 'probability of being open within a burst' is discussed. The single-channel data that we present in this paper support further the idea that the point mutation T671A in the NR2A NMDA receptor subunit affects mainly the ability of glutamate to remain bound to these channels.

Algorithms↗
PubMed2000

Single channel analysis of a novel NMDA channel from Xenopus oocytes expressing recombinant NR1a, NR2A and NR2D subunits.

Two types of subunit (an NR1 subunit and one of the NR2 subunits) are sufficient to form efficient NMDA receptors. In order to investigate whether functional receptors may contain more than one sort of NR2 subunit, NR1, NR2A and NR2D subunits were co-expressed in Xenopus oocytes. Single channel recordings from the oocytes showed, as expected, channel openings that are characteristic of NR1/NR2A receptors (ca 50 and 40 pS conductances), and channels characteristic of NR1/NR2D receptors (ca 40 and 20 pS conductances). However, they also contained a novel channel that contained conductances of ca 30, 40 and 50 pS, with direct transitions between all three levels. It is postulated that the novel channel contains NR1, NR2A and NR2D subunits. The implications of this for receptor stoichiometry are discussed. The novel channel was intermediate between the NR1/NR2A and NR1/NR2D 'duplet' receptors in the length of the channel activations, and in the probability of being open during activation. Its glycine sensitivity was much higher than that of NR1/NR2A, and was comparable with that of the other 'duplet' receptors.

Animals↗
PubMed2000

Issues in managing hyperlipidaemia.

BACKGROUND: The relationship of plasma cholesterol to human coronary artery disease was established with the Framingham study in 1960 and an elevated plasma cholesterol is now considered the major modifiable risk factor for developing coronary artery disease. Recent studies, including the Australian LIPID study, have changed what we consider to be acceptable cholesterol levels. OBJECTIVE: To discuss the role of serum cholesterol levels, dietary modification and lipid lowering drug treatments in overall cardiovascular risk management. DISCUSSION: The current challenges in lipid management are to find ways to increase adherence to medication and deciding how far cholesterol should be lowered. Treatment of a particular patient depends not just on their serum cholesterol but on their overall cardiovascular risk. Diet has cardioprotective effects separate from modification of serum cholesterol levels and this may be true of some lipid lowering treatments.

Coronary Disease↗
PubMed1998

Single-channel activations and concentration jumps: comparison of recombinant NR1a/NR2A and NR1a/NR2D NMDA receptors.

1. We have expressed recombinant NR1a/NR2A and NR1a/NR2D N-methyl-D-aspartate (NMDA) receptor channels in Xenopus oocytes and made recordings of single-channel and macroscopic currents in outside-out membrane patches. For each receptor type we measured (a) the individual single-channel activations evoked by low glutamate concentrations in steady-state recordings, and (b) the macroscopic responses elicited by brief concentration jumps with high agonist concentrations, and we explore the relationship between these two sorts of observation. 2. Low concentration (5-100 nM) steady-state recordings of NR1a/NR2A and NR1a/NR2D single-channel activity generated shut-time distributions that were best fitted with a mixture of five and six exponential components, respectively. Individual activations of either receptor type were resolved as bursts of openings, which we refer to as 'super-clusters'. 3. During a single activation, NR1a/NR2A receptors were open for 36 % of the time, but NR1a/NR2D receptors were open for only 4 % of the time. For both, distributions of super-cluster durations were best fitted with a mixture of six exponential components. Their overall mean durations were 35.8 and 1602 ms, respectively. 4. Steady-state super-clusters were aligned on their first openings and averaged. The average was well fitted by a sum of exponentials with time constants taken from fits to super-cluster length distributions. It is shown that this is what would be expected for a channel that shows simple Markovian behaviour. 5. The current through NR1a/NR2A channels following a concentration jump from zero to 1 mM glutamate for 1 ms was well fitted by three exponential components with time constants of 13 ms (rising phase), 70 ms and 350 ms (decaying phase). Similar concentration jumps on NR1a/NR2D channels were well fitted by two exponentials with means of 45 ms (rising phase) and 4408 ms (decaying phase) components. During prolonged exposure to glutamate, NR1a/NR2A channels desensitized with a time constant of 649 ms, while NR1a/NR2D channels exhibited no apparent desensitization. 6. We show that under certain conditions, the time constants for the macroscopic jump response should be the same as those for the distribution of super-cluster lengths, though the resolution of the latter is so much greater that it cannot be expected that all the components will be resolvable in a macroscopic current. Good agreement was found for jumps on NR1a/NR2D receptors, and for some jump experiments on NR1a/NR2A. However, the latter were rather variable and some were slower than predicted. Slow decays were associated with patches that had large currents.

Animals↗
PubMed1998

A reporter mutation approach shows incorporation of the "orphan" subunit beta3 into a functional nicotinic receptor.

We have investigated whether the neuronal nicotinic subunit beta3 can participate in the assembly of functional recombinant receptors. Although beta3 is expressed in several areas of the central nervous system, it does not form functional receptors when expressed heterologously together with an alpha or another beta nicotinic subunit. We inserted into the human beta3 subunit a reporter mutation (V273T), which, if incorporated into a functional receptor, would be expected to increase its agonist sensitivity and maximum response to partial agonists. Expressing the mutant beta3(V273T) in Xenopus oocytes together with both the alpha3 and the beta4 subunits resulted in the predicted changes in the properties of the resulting nicotinic receptor when compared with those of alpha3 beta4 receptors. This indicated that some of the receptors incorporated the mutant beta3 subunit, as part of a "triplet" alpha3 beta4 beta3 receptor. The proportion of triplet receptors was dependent on the ratios of the alpha3:beta4:beta3 cRNA injected. We conclude that, like the related alpha5 subunit, the beta3 subunit can form functional receptors only if expressed together with both alpha and beta subunits.

Animals↗
PubMed1998

Properties of human glycine receptors containing the hyperekplexia mutation alpha1(K276E), expressed in Xenopus oocytes.

1. Inherited defects in human glycine receptors give rise to hyperekplexia (startle disease). We expressed human glycine receptors in Xenopus oocytes, in order to examine the pharmacological and single-channel properties of receptors that contain a mutation, alpha1(K276E), associated with an atypical form of hyperekplexia. 2. Equilibrium concentration-response curves showed that recombinant human alpha1(K276E)beta receptors had a 29-fold lower glycine sensitivity than wild-type alpha1beta receptors, and a greatly reduced Hill coefficient. The maximum response to glycine also appeared much reduced, whereas the equilibrium constant for the glycine receptor antagonist strychnine was unchanged. 3. Both wild-type and mutant channels opened to multiple conductance levels with similar main conductance levels (33 pS) and weighted mean conductances (41.5 versus 49.8 pS, respectively). 4. Channel openings were shorter for the alpha1(K276E)beta mutant than for the wild-type alpha1beta, with mean overall apparent open times of 0.82 and 6.85 ms, respectively. 5. The main effect of the alpha1(K276E) mutation is to impair the opening of the channel rather than the binding of glycine. This is shown by the results of fitting glycine dose-response curves with particular postulated mechanisms, the shorter open times of mutant channels, the properties of single-channel bursts, and the lack of an effect of the mutation on the strychnine-binding site.

Animals↗
PubMed1998

Identification of amino acid residues of the NR2A subunit that control glutamate potency in recombinant NR1/NR2A NMDA receptors.

The NMDA type of ligand-gated glutamate receptor requires the presence of both glutamate and glycine for gating. These receptors are hetero-oligomers of NR1 and NR2 subunits. Previously it was thought that the binding sites for glycine and glutamate were formed by residues on the NR1 subunit. Indeed, it has been shown that the effects of glycine are controlled by residues on the NR1 subunit, and a "Venus flytrap" model for the glycine binding site has been suggested by analogy with bacterial periplasmic amino acid binding proteins. By analysis of 10 mutant NMDA receptors, we now show that residues on the NR2A subunit control glutamate potency in recombinant NR1/NR2A receptors, without affecting glycine potency. Furthermore, we provide evidence that, at least for some mutated residues, the reduced potency of glutamate cannot be explained by alteration of gating but has to be caused primarily by impairing the binding of the agonist to the resting state of the receptor. One NR2A mutant, NR2A(T671A), had an EC50 for glutamate 1000-fold greater than wild type and a 255-fold reduced affinity for APV, yet it had single-channel openings very similar to those of wild type. Therefore we propose that the glutamate binding site is located on NR2 subunits and (taking our data together with previous work) is not on the NR1 subunit. Our data further imply that each NMDA receptor subunit possesses a binding site for an agonist (glutamate or glycine).

Amino Acid Sequence↗
PubMed1997

The ion channel properties of a rat recombinant neuronal nicotinic receptor are dependent on the host cell type.

1. A stable mammalian cell line (L-alpha 3 beta 4) has been established which expresses the cloned rat neuronal nicotinic acetylcholine receptor (nAChR) subunits alpha 3 and beta 4, which are the most abundant in autonomic ganglia. Ion channel properties of nAChRs expressed in L-alpha 3 beta 4 cells were investigated by single-channel and whole-cell recording techniques, and compared with both rat alpha 3 beta 4 nAChRs expressed in Xenopus oocytes, and endogenous nicotinic receptors in rat superior cervical ganglion (SCG) neurones, using identical solutions for all cell types. 2. Acetylcholine (ACh) caused activation of single ion channel currents with a range of amplitudes. Some channels had high conductances (30-40 pS), and relatively brief lifetimes; these resembled the predominant native channel from SCG. Other channels had low conductances (20-26 pS) and long bursts of openings which were quite unlike native channels, but which were similar to channels formed by alpha 3 beta 4 in oocytes. Both types often occurred in the same patch. 3. Cytisine was about 3 times more potent than ACh (low-concentration potency ratio) in L-alpha 3 beta 4 cells, which is not dissimilar to the 5-fold potency ratio found in both SCG and oocytes, whereas 1,1-dimethyl-4-phenylpiperazinium (DMPP) was less potent than ACh in some cells (as in the oocyte), but more potent in others (as in SCG). 4. While the channels expressed in L-alpha 3 beta 4 cells do not mimic exactly those expressed in rat SCG, they differ considerably from the same subunit combination expressed in oocytes. Larger conductance, SCG-like channels were detected frequently in L-alpha 3 beta 4, but were rarely, if ever, seen in oocytes injected with alpha 3 and beta 4 mRNA. Our results indicate that ion channel properties such as single-channel conductance can be influenced by the choice of heterologous expression system.

Acetylcholine↗