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Receptor-operated ionic channels in the central nervous system during development

The molecular mechanisms underlying the regulation of neurotransmitter-activated ionic channels were analysed by 6 laboratories using a combination of biophysical and biochemical methods.

The project was developed in two main directions: 1. Molecular mechanisms underlying the receptor-operated channel modulation by second messengers (Ca2+, protein phosphorylation). 2. Functional properties of receptor-operated channels during development.

The mechanisms of Ca2+-induced modulation of receptor-operated channels (for acetylcholine (ACh) and NMDA) were studied on mammalian neurons in the central nervous system ( hippocampal slices and tissue culture of cerebellum), on invertebrate neurons (snail Lymnaea stagnalis) and also on human embryonic kidney cell (HEK 293) expressing NMDA and AMPA subtypes of glutamate receptor channels.

On Lymnaea stagnalis neurones elevation of intracellular Ca2+ induced by Ca2+ influx through voltage-activated channels transiently inhibited ACh receptor channels. Sr2+ mimicked the effects of Ca2+ but Ba2+ failed to inhibit ACh receptors. The kinetics of recovery from Ca2+-induced inactivation was in agreement with the kinetics of Ca2+ transients in submembrane layer of the neurones after action potential generation.

Analysis of Ca2+-induced inactivation of NMDA receptor channels was conducted on hippocampal and cerebellar granule neurons using patch-clamp method and confocal scanning microscopy. Double patch-clamp recordings from pairs of rat hippocampal neurons in culture provide a direct evidence that the synaptic NMDAR-channels can be inactivated by rise in (Ca2+)i via dendritic voltage-gated calcium channels. Data obtained on cerebellar granule neurons suggest that Ca-binding protein, calmodulin, is involved in the process of Ca2+-induced inactivation of NMDA receptor channels.

Results on HEK 293 cells show that both NR1-2A and NR1-2B recombinant NMDA receptor channels expressed in HEK 293 cells can be transiently inhibited by Ca2+ ions. Using co-expression of NMDA and AMPA receptor channels in HEK cells we demonstrated that Ca2+ inactivates NMDARs in the time range of excitatory postsynaptic currents (15-25 ms) and a target of Ca2+-induced inactivation is located at the intracellularly located C-terminal domain of the NR1- subunit. These results demonstrate that Ca-induced inactivation of NMDAR-channels is a fast process and it may play an important role in the negative regulation of synaptic transmission.

The pharmacological and biophysical analysis of two main inhibitory neurotransmitters in CNS, GABA and glycine, revealed that post synaptic inhibition changes during postnatal development.
Finally, Ca/calmodulin-dependent protein kinase 2 (CAMK2) in synaptic membranes from the brain of ground squirrels at different stages of hibernation was found to be mainly in autophosphorylated for opposite to CAMK2 from the brain of rats. It is suggested that this feature facilitates switching between active and torpid states of animals. 32P incorporation into 56 kDa, 58 kDa and 53 kDa proteins from brain of ground squirrels was shown to depend on their functional states. The dependence of 53 kDa protein phosphorylation was eliminated by oleic acid treatment of synaptic membranes that might reflect the important role of lipid composition of membranes in the adaption of mammals to low temperature.

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