Allosteric regulation of synaptic processes

Abstract

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.