Studies of NMDA receptor function and stoichiometry with truncated and tandem subunits

The subunits that compose eukaryotic glutamate ion channel receptors have three transmembrane domains (TMs) and terminate with intracellular tails that are important for controlling channel expression and localization. Truncation of NMDA receptor subunits before the final TM showed that this TM and intracellular tail region are necessary to form functional channels. However, it is shown here that these truncated subunits may be partially rescued by coexpressing the final TM and tail as a separate protein. The whole-cell currents so produced are somewhat lower than with full-length subunits, and they do not show the sag characteristic of currents from channels containing NR1 and NR2A subunits in the continued presence of an agonist. In addition, these truncated subunits were joined to full-length subunits to generate tandems. The functional expression of these tandems confirmed the tetrameric structure of NMDA receptors and also suggested that the subunits making up NMDA receptors are arranged as a dimer of dimers in the receptors with a 1-1-2-2 orientation of the subunits in the channel, and not in an alternating pattern of subunits around the pore. These results may redirect future studies into the mechanism of binding and gating in these receptors toward schemes including dimers, and may also be relevant to studies of glutamate receptor ion channels in general

The reproducibility of research and the misinterpretation of p-values

We wish to answer this question: If you observe a ‘significant’ p-value after doing a single unbiased experiment, what is the probability that your result is a false positive? The weak evidence provided by p-values between 0.01 and 0.05 is explored by exact calculations of false positive risks. When you observe p = 0.05, the odds in favour of there being a real effect (given by the likelihood ratio) are about 3 : 1. This is far weaker evidence than the odds of 19 to 1 that might, wrongly, be inferred from the p-value. And if you want to limit the false positive risk to 5%, you would have to assume that you were 87% sure that there was a real effect before the experiment was done. If you observe p = 0.001 in a well-powered experiment, it gives a likelihood ratio of almost 100 : 1 odds on there being a real effect. That would usually be regarded as conclusive. But the false positive risk would still be 8% if the prior probability of a real effect were only 0.1. And, in this case, if you wanted to achieve a false positive risk of 5% you would need to observe p = 0.00045. It is recommended that the terms ‘significant’ and ‘non-significant’ should never be used. Rather, p-values should be supplemented by specifying the prior probability that would be needed to produce a specified (e.g. 5%) false positive risk. It may also be helpful to specify the minimum false positive risk associated with the observed p-value. Despite decades of warnings, many areas of science still insist on labelling a result of p < 0.05 as ‘statistically significant’. This practice must contribute to the lack of reproducibility in some areas of science. This is before you get to the many other well-known problems, like multiple comparisons, lack of randomization and p-hacking. Precise inductive inference is impossible and replication is the only way to be sure. Science is endangered by statistical misunderstanding, and by senior people who impose perverse incentives on scientists

How to treat hypercholesterolaemia

An elevated low density lipoprotein cholesterol is a major cause of atherosclerosis. Reducing the concentration of this lipoprotein stabilises atherosclerotic plaques, and may lead to regression of the atherosclerosis. A moderate reduction of the plasma concentration of this lipoprotein significantly decreases recurrent coronary events. Therapy is a combination of lifestyle modification, nutraceuticals and drug treatment. The most convenient and effective drugs are the HMGCoA reductase inhibitors or 'statins'. They control hyperlipidaemia and help to prevent myocardial infarction, unstable angina, sudden death and stroke

Direct iminization of PEEK

Semi-crystalline poly(ether ketone)s are important high-temperature engineering thermoplastics, but are difficult to characterize at the molecular level because of their insolubility in conventional organic solvents. Here we report that polymers of this type, including PEEK, react cleanly at high temperatures with low-volatility aralkyl amines to afford stable, noncrystalline poly(ether-imine)s, which are readily soluble in solvents such as chloroform, THF and DMF and so characterizable by conventional size-exclusion chromatography

The activation mechanism of alpha 1 homomeric glycine receptors

The glycine receptor mediates fast synaptic inhibition in the spinal cord and brainstem. Its activation mechanism is not known, despite the physiological importance of this receptor and the fact that it can serve as a prototype for other homopentameric channels. We analyzed single-channel recordings from rat recombinant alpha1 glycine receptors by fitting different mechanisms simultaneously to sets of sequences of openings at four glycine concentrations (10-1000 muM). The adequacy of the mechanism and the rate constants thus fitted was judged by examining how well these described the observed dwell-time distributions, open-shut correlation, and single-channel P-open dose-response curve. We found that gating efficacy increased as more glycine molecules bind to the channel, but maximum efficacy was reached when only three (of five) potential binding sites are occupied. Successive binding steps are not identical, implying that binding sites can interact while the channel is shut. These interactions can be interpreted in the light of the topology of the binding sites within a homopentamer

Openings of the rat recombinant alpha1 homomeric glycine receptor as a function of the number of sgonist molecules bound

The functional properties of rat homomeric {alpha}1 glycine receptors were investigated using whole-cell and outside-out recording from human embryonic kidney cells transfected with rat {alpha}1 subunit cDNA. Whole-cell dose-response curves gave EC50 estimates between 30 and 120 µM and a Hill slope of ~3.3. Single channel recordings were obtained by steady-state application of glycine (0.3, 1, or 10 µM) to outside-out patches. Single channel conductances were mostly 60–90 pS, but smaller conductances of ~40 pS were also seen (10% of the events) with a relative frequency that did not depend on agonist concentration. The time constants of the apparent open time distributions did not vary with agonist concentration, but short events were more frequent at low glycine concentrations. There was also evidence of a previously missed short-lived open state that was more common at lower glycine concentrations. The time constants for the different components of the burst length distributions were found to have similar values at different concentrations. Nevertheless, the mean burst length increased with increasing glycine. This was because the relative area of each burst-length component was concentration dependent and short bursts were favored at lower glycine concentrations. Durations of adjacent open and shut times were found to be strongly (negatively) correlated. Additionally, long bursts were made up of longer than average openings separated by short gaps, whereas short bursts usually consisted of single isolated short openings. The most plausible explanation for these findings is that long bursts are generated when a higher proportion of the five potential agonist binding sites on the receptor is occupied by glycine. On the basis of the concentration dependence and the intraburst structure we provide a preliminary kinetic scheme for the activation of the homomeric glycine receptor, in which any number of glycine molecules from one to five can open the channel, although not with equal efficiency

Single channel study of the spasmodic mutation α1A52S in recombinant rat glycine receptors

Inherited defects in glycine receptors lead to hyperekplexia, or startle disease. A mutant mouse, spasmodic, that has a startle phenotype, has a point mutation (A52S) in the glycine receptor α1 subunit. This mutation reduces the sensitivity of the receptor to glycine, but the mechanism by which this occurs is not known. We investigated the properties of A52S recombinant receptors by cell-attached patch clamp recording of single-channel currents elicited by 30 – 10000 μM glycine. We used heteromeric receptors, which resemble those found at adult inhibitory synapses. Activation mechanisms were fitted directly to single channel data using the HJCFIT method, which includes an exact correction for missed events. In common with wildtype receptors, only mechanisms with three binding sites and extra shut states could describe the observations. The most physically plausible of these, the ‘flip’ mechanism, suggests that pre-opening isomerisation to the flipped conformation that follows binding is less favoured in mutant than in wild-type receptors, and, especially, that the flipped conformation has a 100-fold lower affinity for glycine than in wildtype receptors. In contrast, the efficacy of the gating reaction was similar to that of wild-type heteromeric receptors. The reduction in affinity for the flipped conformation accounts for the reduction in apparent cooperativity seen in the mutant receptor (without having to postulate interaction between the binding sites) and it accounts for the increased EC50 for responses to glycine that is seen in mutant receptors. This mechanism also predicts accurately the faster decay of synaptic currents that is observed in spasmodic mice

Syntheses, Hydrogen Bonding, and Metal Coordination Studies of New Ferrocene Peptide Conjugates

The main goal of the research was the synthesis of new ferrocene peptideconjugates, to study the role of hydrogen bonding interactions, and the coordinationproperties of these systems. Ferrocene peptide conjugates have been extensivelystudied as models of protein secondary structures. Of particular interest weredisubstituted ferrocene derivatives such as ferrocene dicarboxylic acid, which have beenshown to be important in the design of peptide mimics. The inter-ring spacing betweentwo cyclopentadienyl rings in ferrocene has shown to be appropriate for forminghydrogen bonds between conjugated peptide chains, which are attached to the twocyclopentadienyl rings. Inter-ring spacing has proven to be of great importance for theformation of stable hydrogen-bonded structures. Additionally, some of these ferrocenepeptide conjugates have the ability to coordinate metal ions. This is of interest becausethese systems have the potential to serve as active site mimics of metalloproteins. Thiswork focused on the synthesis, hydrogen bonding studies, and coordination propertiesof novel ferrocene peptide conjugates

Temperature and inoculation method influence disease phenotypes and mortality of Eucalyptus marginata clonal lines inoculated with Phytophthora cinnamomi

Survival of 1-year-old plants of three clonal lines of Eucalyptus marginata (jarrah), two ranked as resistant (RR1 and RR2) and one as susceptible (SS1) to Phytophthora cinnamomi, was assessed after pathogen inoculation with either mycelial mats underbark or zoospores on the stem. Plants were grown at 15, 20, 25 and 30°C. Method of inoculation did not produce comparable mortalities of the clonal lines, particularly at 25 and 30°C. At these temperatures, all three clonal lines had 100% mortality when inoculated underbark, but when inoculated with zoospores, RR1 had 60% survival and lines SS1 and RR2 had 100% mortality. Generally, the level of resistance of all clonal lines declined with increasing temperature. RR2 had consistently higher mortality than SS1, and is therefore not considered resistant. Lesion development was also measured in detached stems of RR1 and a susceptible clonal line (SS2) each inoculated underbark with four different P. cinnamomi isolates. Stems were assessed for lesion development at 20, 25 and 30°C for 4 days. For all four isolates, detached stems of RR1 generally had smaller lesions than those of SS2, particularly at 30°C. The increase in lesion length with increasing temperature was greatest for SS2. Detached stems may have potential in screening for jarrah resistant toP. cinnamomi and allow identification of susceptible clonal lines at 30°C