Allosteric regulation of synaptic processes

Glutamatergic neurotransmission is of key importance for short-term and long-term plasticity in the hippocampus, a part of the medial temporal lobe which is responsible for processes of explicit semantic and spatial memory. Short-term plasticity is mainly regulated by the presynaptic neuron and long-term plasticity is to large parts regulated by the post-synaptic neuron. In this thesis we have looked into cellular and molecular biophysical mechanisms in glutamatergic neurons mainly in the hippocampus. We first reviewed the presynaptic mechanisms underlying short-term plasticity like assembly of the release machinery, positional and molecular priming, site preparation, calcium dynamics regulation, intrinsic vesicular fusogenicity, endocytosis, acidification and filling. In study 1 we looked into the role of intrinsic vesicle fusogenicity on short-term plasticity by formulating a deterministic vesicular release model based on ordinary differential equations. Intrinsic vesicular fusogenicity was an allosteric property we invented in order to test the hypothesis of calcium independence. The model was able to simulate properties of resting neurons, by reproducing the spontaneous release rates and the size of the readily releasable pool. Furthermore, assuming that the heterogeneity in vesicular release probability arises due to differences in intrinsic vesicular fusogenicity, the model was able to explain depression by an imbalance between fusion and vesicular priming. It also predicted that facilitation could be due to an increase in intrinsic vesicular fusogenicity, which together with build-up of calcium gave rise to initial increase in vesicular release. Finally, we investigated the effect of three different modes of regulation of release probability on short-term plasticity. It was seen that differences in intrinsic vesicular fusogenicity gave rise to a more significant change in shortterm plasticity than change in calcium sensitivity of release. All in all the results tell us that intrinsic vesicular fusogenicity has an important role in tuning short-term plasticity. In study 2 we investigated the regulation of the postsynaptic allosteric AMPA receptor. To do this we developed a model based on the Monod Wyman Changeux framework which described the ligand concentration dependence of the conductance states by increasing affinity to conductance states. The model was able to explain thermodynamic behaviours of native and recombinant receptors when stimulated with full agonists like glutamate and quisqualate as well as partial agonists like willardiines. It was also predicted that the receptor stabilizes its large conductance state within the rise time of a so-called 'mini' post-synaptic current, providing a possible underlying mechanism for the peak of the current. In study 3 we investigated the high-dose hook effect in allosteric proteins by first developing a combinatorical theory for how linker proteins behave under conditions of perfect binding. The theory predicted that the steady-state concentration of fully bound linker-proteins decreases at a critical concentration of initial free linker protein as the free linker protein concentration is increased. This effect is however decreased in proteins where binding of ligand occurs in a cooperative fashion. The outcome was validated by simulations of dimeric and tetrameric linker proteins under imperfect binding. We also simulated the cooperative synaptic protein calmodulin, and it was seen to be subject to the hook effect. The hook effect was stronger in the presence of the allosteric activator Ca2+/calmodulin kinase II (CamKII). We show that increased amounts of the allosteric activator can decrease the activity of calmodulin. At 140 uM calmodulin behaved only as if the molecule only appeared in the relaxed (R) state. The relaxed state has no cooperativity, but has higher ligand affinity than the wild-type calmodulin. Even though this phenomenon may be present in many different biochemical systems, synapses contain several linker proteins that are pivotal for synaptic plasticity for instance AMPA receptors, synaptotagmin, calbinding and calmodulin. In summary, this thesis gives insight into allosteric mechanisms in glutamatergic hippocampal neurons by using whole-cell voltage clamp and algebraic modelling. Specifically, it suggests an explanation for the important role of allosteric mechanisms in vesicular release probability and short-term plasticity. It also provides an explanation for the ligand concentration dependence of AMPA receptors and puts forward a theory for how complexes and active forms of linker proteins behave under increase of free linker protein concentration, a behaviour might contribute to pre-and postsynaptic processes

Ligand-dependent opening of the multiple AMPA receptor conductance states: a concerted model

Modulation of the properties of AMPA receptors at the post-synaptic membrane is one of the main suggested mechanisms behind synaptic plasticity in the central nervous system of vertebrates. Electrophysiological recordings of single channels stimulated with agonists showed that both recombinant and native AMPA receptors visit multiple conductance states in an agonist concentration dependent manner. We propose an allosteric model of the multiple conductance states based on concerted conformational transitions of the four subunits, as an iris diaphragm. Our model predicts that the thermodynamic behaviour of the conductance states upon full and partial agonist stimulations can be described with increased affinity of receptors as they progress to higher conductance states. The model also predicts existence of AMPA receptors in non-liganded conductive substates. However, spontaneous openings probability decreases with increasing conductances. Finally, we predict that the large conductance states are stabilized within the rise phase of a whole-cell EPSC in glutamatergic hippocampal neurons. Our model provides a mechanistic link between ligand concentration and conductance states that can explain thermodynamic and kinetic features of AMPA receptor gating.Comment: 4 figures, models available on demand. They will be published by BioModels Database upon publication of the articl

Biophysical properties of presynaptic short-term plasticity in hippocampal neurons: insights from electrophysiology, imaging and mechanistic models

Hippocampal neurons show different types of short-term plasticity (STP). Some exhibit facilitation of their synaptic responses and others depression. In this review we discuss presynaptic biophysical properties behind heterogeneity in STP in hippocampal neurons such as alterations in vesicle priming and docking, fusion, neurotransmitter filling and vesicle replenishment. We look into what types of information electrophysiology, imaging and mechanistic models have given about the time scales and relative impact of the different properties on STP with an emphasis on the use of mechanistic models as complementary tools to experimental procedures. Taken together this tells us that it is possible for a multitude of different mechanisms to underlie the same STP pattern, even though some are more important in specific cases, and that mechanistic models can be used to integrate the biophysical properties to see which mechanisms are more important in specific cases of STP

Cooperative binding mitigates the high-dose hook effect

Background: The high-dose hook effect (also called prozone effect) refers to the observation that if a multivalent protein acts as a linker between two parts of a protein complex, then increasing the amount of linker protein in the mixture does not always increase the amount of fully formed complex. On the contrary, at a high enough concentration range the amount of fully formed complex actually decreases. It has been observed that allosterically regulated proteins seem less susceptible to this effect. The aim of this study was two-fold: First, to investigate the mathematical basis of how allostery mitigates the prozone effect. And second, to explore the consequences of allostery and the high-dose hook effect using the example of calmodulin, a calcium-sensing protein that regulates the switch between long-term potentiation and long-term depression in neurons. Results: We use a combinatorial model of a “perfect linker protein” (with infinite binding affinity) to mathematically describe the hook effect and its behaviour under allosteric conditions. We show that allosteric regulation does indeed mitigate the high-dose hook effect. We then turn to calmodulin as a real-life example of an allosteric protein. Using kinetic simulations, we show that calmodulin is indeed subject to a hook effect. We also show that this effect is stronger in the presence of the allosteric activator Ca 2+/calmodulin-dependent kinase II (CaMKII), because it reduces the overall cooperativity of the calcium-calmodulin system. It follows that, surprisingly, there are conditions where increased amounts of allosteric activator actually decrease the activity of a protein. Conclusions: We show that cooperative binding can indeed act as a protective mechanism against the hook effect. This will have implications in vivo where the extent of cooperativity of a protein can be modulated, for instance, by allosteric activators or inhibitors. This can result in counterintuitive effects of decreased activity with increased concentrations of both the allosteric protein itself and its allosteric activators. Electronic supplementary material The online version of this article (doi:10.1186/s12918-017-0447-8) contains supplementary material, which is available to authorized users

Allosteric and dissociation constants determined for full agonists.

<p>Allosteric and dissociation constants determined for full agonists.</p

Dissociation constants for partial agonists.

<p>Dissociation constants for partial agonists.</p

Kinetic behaviour of synaptic AMPARs.

<p>(<b>A</b>) The blue trace shows the average synaptic current, which reaches its peak within fractions of ms (n = 7). The black bar represents the depolarisation of the pre-synapic terminal. (<b>B</b>) Kinetics of the subconductance states of an AMPA receptor population (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116616#pone.0116616.s005" target="_blank">S4 Supporting model</a>). Left plot, deterministic simulation of a population of GluA3/GluK2 receptors by 1 μM of agonist. Right plot, stochastic simulation of a population of 50 receptors. Only the most populated states are represented for sake of clarity.</p

Effects of full and partial agonists.

<p>(<b>A</b>) shows the small, medium and large conductance states upon stimulation with glutamate where the large state is stabilized (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116616#pone.0116616.s002" target="_blank">S1 Supporting model</a>). The relative frequency of the small conductance state (green line) is 0.6 at a ligand concentration of 1 μM and decreases when the ligand concentration is increased, whereas the medium conductance state (blue line) reaches its peak at a concentration above 0.1 μM and most receptors are found in the large conductance state (black line) at 10 μM. The dots represent experimental data [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116616#pone.0116616.ref015" target="_blank">15</a>]. (<b>B</b>) stabilization of GluA3/GluK2 receptor large conductance state upon stimulation with quisqualate [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116616#pone.0116616.ref002" target="_blank">2</a>] (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116616#pone.0116616.s003" target="_blank">S2 Supporting model</a>). The relative frequencies of the basal state (red line) and small state (green line) of the receptor are 0.7 and 0.25 at 1 μM and decrease when ligand concentration is increased, whereas the medium (blue) and large (black) conductance states increase and reach 0.1 and 0.9 respectively at 1 mM. The line in magenta shows the saturation function. (<b>C</b>) stabilization of GluR2 homomeric receptors intermediate conductance state upon stimulation with large willardiines (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116616#pone.0116616.s004" target="_blank">S3 Supporting model</a>). The relative frequencies of the small state (green lines) is decreased and the medium (blue) and large (black) states are increased when the ligand concentration increases at stimulation with both BrW and IW. At a ligand concentration of 10 mM the relative frequency of the medium state was 0.65 and 0.35 at stimulation with IW (dashed) and BrW (solid) respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116616#pone.0116616.ref010" target="_blank">10</a>].</p

State transitions of a single channel.

<p>A single channel progresses from a non-liganded basal state to a fully liganded large open state within 0.4 ms upon stimulation with 1 μM full agonist (the original STOIC model is provided as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116616#pone.0116616.s005" target="_blank">S4 Supporting model</a>). It should be noted that the simulation is stochastic and this is one of the possible paths the receptor takes to its stable state.</p