Energetics of Shaker K channels block by inactivation peptides

A synthetic peptide of the NH2-terminal inactivation domain of the ShB channel blocks Shaker channels which have an NH2-terminal deletion and mimics many of the characteristics of the intramolecular inactivation reaction. To investigate the role of electrostatic interactions in both peptide block and the inactivation process we measured the kinetics of block of macroscopic currents recorded from the intact ShB channel, and from ShB delta 6-46 channels in the presence of peptides, at different ionic strengths. The rate of inactivation and the association rate constants (k(on)) for the ShB peptides decreased with increasing ionic strength. k(on) for a more positively charged peptide was more steeply dependent on ionic strength consistent with a simple electrostatic mechanism of enhanced diffusion. This suggests that a rate limiting step in the inactivation process is the diffusion of the NH2-terminal domain towards the pore. The dissociation rates (k(off)) were insensitive to ionic strength. The temperature dependence of k(on) for the ShB peptide was very high, (Q10 = 5.0 +/- 0.58), whereas k(off) was relatively temperature insensitive (Q10 approximately 1.1). The results suggest that at higher temperatures the proportion of time either the peptide or channel spends in the correct conformation for binding is increased. There were two components to the time course of recovery from block by the ShB peptide, indicating two distinct blocked states, one of which has similar kinetics and dependence on external K+ concentration as the inactivated state of ShB. The other is voltage-dependent and at -120 mV is very unstable. Increasing the net charge on the peptide did not increase sensitivity to knock-off by external K+. We propose that the free peptide, having fewer constraints than the tethered NH2-terminal domain binds to a similar site on the channel in at least two different conformations

The trafficking and targeting of P2X receptors

The functional expression of P2X receptors at the plasma membrane is dependent on their trafficking along secretory and endocytic pathways. There are seven P2X receptor subunits, and these differ in their subcellular distributions because they have very different trafficking properties. Some are retained within the endoplasmic reticulum (ER), while others are predominantly at the cell surface or within endosomes and lysosomes. Changes in recruitment of receptors to and from the plasma membrane provides a way of rapidly up- or down-regulating the cellular response to adenosine triphosphate (ATP). An additional layer of regulation is the targeting of these receptors within the membranes of each compartment, which affects their stability, function and the nature of the effector proteins with which they form signaling complexes. The trafficking and targeting of P2X receptors is regulated by their interactions with other proteins and with lipids and we can expect this to vary in a cell-type specific manner and in response to changes in the environment giving rise to differences in receptor activity and function

Interactions of amino terminal domains of Shaker K channels with a pore blocking site studied with synthetic peptides

Synthetic peptides of the five alternative NH2-terminal sequences of Shaker when applied to the cytoplasmic side of ShB channels that have an NH2-terminal deletion (ShB delta 6-46) block the channel with potencies correlated with the rate of inactivation in the corresponding variant. These peptides share no sequence similarity and yet three out of the five have apparent dissociation constants between 2 and 15 microM, suggesting that the specificity requirements for binding are low. To identify the primary structural determinants required for effective block of ShB delta 6-46, we examined the effects of substitutions made to the 20 residue ShB peptide on association and dissociation rates. Nonpolar residues within the peptide appear to be important in stabilizing the binding through hydrophobic interactions. Substitutions to leucine-7 showed there was a clear correlation between hydrophobicity and the dissociation rate constant (koff) with little effect on the association rate constant (kon). Substituting charged residues for hydrophobic residues within the region 4-8 disrupted binding. Within the COOH-terminal half of the peptide, substitutions that increased the net positive charge increased kon with relatively small changes in koff, suggesting the involvement of long-range electrostatic interactions in increasing the effective concentration of the peptide. Neutralizing charged residues produced small changes in koff. Charges within the region 12-20 act equivalently; alterations which conserved net charge produced little effect on either kon or koff. The results are consistent with this region of the peptide having an extended conformation and suggest that when bound this region makes few contacts with the channel protein and remains relatively unconstrained. Analogous mutations within the NH2-terminal domain of the intact ShB channel produced qualitatively similar effects on blocking and unblocking rates

Are Jurors Intuitive Statisticians? Bayesian Causal Reasoning in Legal Contexts

In criminal trials, evidence often involves a degree of uncertainty and decision-making includes moving from the initial presumption of innocence to inference about guilt based on that evidence. The jurors’ ability to combine evidence and make accurate intuitive probabilistic judgments underpins this process. Previous research has shown that errors in probabilistic reasoning can be explained by a misalignment of the evidence presented with the intuitive causal models that people construct. This has been explored in abstract and context-free situations. However, less is known about how people interpret evidence in context-rich situations such as legal cases. The present study examined participants’ intuitive probabilistic reasoning in legal contexts and assessed how people’s causal models underlie the process of belief updating in the light of new evidence. The study assessed whether participants update beliefs in line with Bayesian norms and if errors in belief updating can be explained by the causal structures underpinning the evidence integration process. The study was based on a recent case in England where a couple was accused of intentionally harming their baby but was eventually exonerated because the child’s symptoms were found to be caused by a rare blood disorder. Participants were presented with a range of evidence, one piece at a time, including physical evidence and reports from experts. Participants made probability judgments about the abuse and disorder as causes of the child’s symptoms. Subjective probability judgments were compared against Bayesian norms. The causal models constructed by participants were also elicited. Results showed that overall participants revised their beliefs appropriately in the right direction based on evidence. However, this revision was done without exact Bayesian computation and errors were observed in estimating the weight of evidence. Errors in probabilistic judgments were partly accounted for, by differences in the causal models representing the evidence. Our findings suggest that understanding causal models that guide people’s judgments may help shed light on errors made in evidence integration and potentially identify ways to address accuracy in judgment

Causal judgments about atypical actions are influenced by agents' epistemic states

A prominent finding in causal cognition research is people’s tendency to attribute increased causality to atypical actions. If two agents jointly cause an outcome (conjunctive causation), but differ in how frequently they have performed the causal action before, people judge the atypically acting agent to have caused the outcome to a greater extent. In this paper, we argue that it is the epistemic state of an abnormally acting agent, rather than the abnormality of their action, that is driving people's causal judgments. Given the predictability of the normally acting agent's behaviour, the abnormal agent is in a better position to foresee the consequences of their action. We put this hypothesis to test in four experiments. In Experiment 1, we show that people judge the atypical agent as more causal than the normally acting agent, but also judge the atypical agent to have an epistemic advantage. In Experiment 2, we find that people do not judge a causal difference if no epistemic advantage for the abnormal agent arises. In Experiment 3, we replicate these findings in a scenario in which the abnormal agent's epistemic advantage generalises to a novel context. In Experiment 4, we extend these findings to mental states more broadly construed and develop a Bayesian network model that predicts the degree of outcome-oriented mental states based on action normality and epistemic states. We find that people infer mental states like desire and intention to a greater extent from abnormal behaviour when this behaviour is accompanied by an epistemic advantage. We discuss these results in light of current theories and research on people's preference for abnormal causes

P2X receptor trafficking in neurons is subunit specific

P2X receptors within the CNS mediate excitatory synaptic transmission and also act presynaptically to modulate neurotransmitter release. We have studied the targeting and trafficking of P2X4 and P2X2 receptors heterologously expressed in cultured olfactory bulb neurons. Homomeric P2X4 receptors had a punctate distribution, and many of the puncta colocalized with early endosomes. In contrast, P2X2 receptors were primarily localized at the plasma membrane. By antibody-labeling of surface receptors in living neurons, we showed that P2X4 receptors undergo rapid constitutive internalization and subsequent reinsertion into the plasma membrane, whereas P2X2 receptors were not regulated in such a way. The internalization of P2X4 receptors was dynamin-dependent, and the binding of ATP enhanced the basal rate of retrieval in a Ca2+-independent manner. The presence of the P2X4 subunit in a P2X4/6 heteromer governed the trafficking properties of the receptor. P2X receptors acted presynaptically to enhance the release of glutamate, suggesting that the regulated cycling of P2X4-containing receptors might provide a mechanism for modulation of synaptic transmission

The stoichiometry of P2X2/6 receptor heteromers depends on relative subunit expression levels

Fast synaptic transmission involves the operation of ionotropic receptors, which are often composed of at least two types of subunit. We have developed a method, based on atomic force microscopy imaging to determine the stoichiometry and subunit arrangement within ionotropic receptors. We showed recently that the P2X(2) receptor for ATP is expressed as a trimer but that the P2X(6) subunit is unable to oligomerize. In this study we addressed the subunit stoichiometry of heteromers containing both P2X(2) and P2X(6) subunits. We transfected tsA 201 cells with both P2X(2) and P2X(6) subunits, bearing different epitope tags. We manipulated the transfection conditions so that either P2X(2) or P2X(6) was the predominant subunit expressed. By atomic force microscopy imaging of isolated receptors decorated with antiepitope antibodies, we demonstrate that when expression of the P2X(2) subunit predominates, the receptors contain primarily 2 x P2X(2) subunits and 1 x P2X(6) subunit. In contrast, when the P2X(6) subunit predominates, the subunit stoichiometry of the receptors is reversed. Our results show that the composition of P2X receptor heteromers is plastic and dependent on the relative subunit expression levels. We suggest that this property of receptor assembly might introduce an additional layer of subtlety into P2X receptor signaling

The propensity interpretation of probability and diagnostic split in explaining away

Causal judgements in explaining-away situations, where multiple independent causes compete to account for a common effect, are ubiquitous in both everyday and specialised contexts. Despite their ubiquity, cognitive psychologists still struggle to understand how people reason in these contexts. Empirical studies have repeatedly found that people tend to ‘insufficiently’ explain away: that is, when one cause explains the presence of an effect, people do not sufficiently reduce the probability of other competing causes. However, the diverse accounts that researchers have proposed to explain this insufficiency suggest we are yet to find a compelling account of these results. In the current research we explored the novel possibility that insufficiency in explaining away is driven by: (i) some people interpreting probabilities as propensities, i.e. as tendencies of a physical system to produce an outcome and (ii) some people splitting the probability space among the causes in diagnostic reasoning, i.e. by following a strategy we call ‘the diagnostic split’. We tested these two hypotheses by manipulating (a) the characteristics of cover stories to reflect different degrees to which the propensity interpretation of probability was pronounced, and (b) the prior probabilities of the causes which entailed different normative amounts of explaining away. Our results were in line with the extant literature as we found insufficient explaining away. However, we also found empirical support for our two hypotheses, suggesting that they are a driving force behind the reported insufficiency

Strategies for selecting and evaluating information

Within the domain of psychology, Optimal Experimental Design (OED) principles have been used to model how people seek and evaluate information. Despite proving valuable as computational-level methods to account for people's behaviour, their descriptive and explanatory powers remain largely unexplored. In a series of experiments, we used a naturalistic crime investigation scenario to examine how people evaluate queries, as well as outcomes, in probabilistic contexts. We aimed to uncover the psychological strategies that people use, not just to assess whether they deviated from OED principles. In addition, we explored the adaptiveness of the identified strategies across both one-shot and stepwise information search tasks. We found that people do not always evaluate queries strictly in OED terms and use distinct strategies, such as by identifying a leading contender at the outset. Moreover, we identified aspects of zero-sum thinking and risk aversion that interact with people's information search strategies. Our findings have implications for building a descriptive account of information seeking and evaluation, accounting for factors that currently lie outside the realm of information-theoretic OED measures, such as context and the learner's own preferences

Updating Prior Beliefs based on Ambiguous Evidence

This paper investigates a problem where the solver must firstly determine which of two possible causes are the source of an effect where one cause has a historically higher propensity to cause that effect. Secondly, they must update the propensity of the two causes to produce the effect in light of the observation. Firstly, we find an error commensurate with the ‘double updating’ error observed within the polarisation literature: individuals appear to first use their prior beliefs to interpret the evidence, then use the interpreted form of the evidence, rather than the raw form, when updating. Secondly, we find an error where individuals convert from a probabilistic representation of the evidence to a categorical one and use this representation when updating. Both errors have the effect of exaggerating the evidence in favour of the solver’s prior belief and could lead to confirmation bias and polarisation