Stochastic model of glycine transporter and its application to amino acid transport in mammalian neurons

Zaytsev Kirill, Boronovskiy Stanislav, Nartsissov Yaroslav
2014 Biochimica et Biophysica Acta - Bioenergetics  
The metabolite exchange between mitochondria and cytoplasm is supported by VDAC (voltage-dependent anion selective channel). Studies concerning the role of VDAC have proved that the channel participates in ATP rationing, Ca 2 + homeostasis, intracellular redox state regulation, communication between mitochondria and nucleus and apoptosis execution. Thus, the channel is regarded as crucial for mitochondria functioning and consequently for cell life or death. In mitochondria of different
more » ... VDAC may be present as isoforms encoded by separated genes, displaying different channel-forming activities and probably playing different roles. Saccharomyces cerevisiae mitochondria express two VDAC isoforms (yVDAC1 and yVDAC2), of which only yVDAC1, encoded by POR1 gene, has been proved to form a channel with properties highly conserved in other species. In human mitochondria, as in the case of other vertebrates, three isoforms of VDAC (hVDAC1-hVDAC3) able to form functional channels have been identified. They are expressed in different tissues and organs at different levels. Huntington's disease (HD) is an autosomal-dominant neurodegenerative hereditary disorder that gradually robs affected individuals of memory, cognitive skills and normal movements. It is originated by the mutation of the gene encoding the huntingtin-protein (Htt). Htt with an abnormal stretch of above 35 glutamines in the N terminus (mHtt) results in HD. The observed symptoms correlate with the selective loss of neurons within the central nervous system, not only in the striatum but also in the cerebral cortex. At present increasing amount of data indicates that mitochondrial functioning is affected by mHtt and the resulting mitochondrial impairments may occur early enough to contribute to mHtt-induced toxicity and the HD pathogenic mechanism. In our studies, we focused on the interaction of hVDAC1-hVDAC3 with Htt and mHtt. Therefore, we examined the effect of GST-Htt exon 1 fusion proteins containing 28 (Htt) and 74 (mHtt) glutamines on channel properties of the VDAC proteins isolated from Δpor1hVDAC1, Δpor1hVDAC2 and Δpor1hVDAC3 S. cerevisiae cells as well as from neuroblastoma cells. Obtained results indicate that Htt and mHtt directly and differently modulate human VDAC. This in turn could be important for development of new therapeutic strategies concerning HD. Acknowledgements: the studies were supported by the grant: NCN 2011/01/B/NZ3/00359. Neurotransmitter uptake is quite essential in various metabolic and functional processes in neural tissue. Inhibitory glycinergic neurotransmission is terminated by specific glycine transporters GlyTs (GlyT1 and GlyT2), which actively reuptake glycine from the synaptic cleft. In physiological conditions the pool of this amino acid is regulated by a cascade of metabolic reactions and by membrane transport via specific transporters as well. The re-uptake of glycine into presynaptic terminals and surrounding glia is obligatory for the maintenance of low synaptic levels of the transmitter in the synaptic cleft. Glycine transporter type 2 (GlyT2) presented in the presynaptic membrane is a member of Na + /Cl − -dependent transport proteins family, which share a common structure with 12 transmembrane domains. Transport of one glycine molecule involves the transport of one Cl − and 3 Na + per transport cycle. The main goal of the present study was to develop a computer simulator of GlyT2 activity based on known experimental data for quantitative estimation of membrane glycine transport. A sequence of elemental events happening during the cycle of GlyT2 activity was summarized as a single scheme, which became a basis of an original software. The algorithm of transporter simulator was developed using the probability approach describing the behavior of a single protein. As a result of such computations the number of translocated glycine molecules per time period has been evaluated. The computer experiments were carried out under different environmental conditions such as ion and glycine concentrations. As the major equilibrium constants of the transport steps are still unmeasured the reversibility degree of the glycine transport is also considered as a variable parameter. Using described software the time dependences of glycine, sodium and chloride ions amounts were obtained. Ligand cooperativity was observed for sodium ions (Hill coefficient is 3.6). The developed software based on proposed probability algorithm can be used for a virtual experiment in GLYT2 activity simulation. The described model allows to predict some characteristics of the transporter functioning which can be experimentally proven. This software combined with glycine receptor model can be also used in research laboratories for evaluating concentrations of chloride, sodium and glycine in synaptic cleft and presynaptic terminal in different time points during inhibitory signaling. Macrovipera lebetina obtusa (MLO) is one of the most important poisonous snakes in Armenia. In the venom of this snake a specific toxin was not identified but they form complexes with other nonenzymatic proteins to achieve higher efficiency through synergy. We have studied influence of venom on the erythrocyte ghosts by fluorescent microscopy. Images were collected on an epi-fluorescent microscope FM320-5M (AmScope, USA). The erythrocyte ghosts were visualized with ANS fluorescent probe. The erythrocyte ghosts were deformed after adding the MLO venom. They shrink within 3 min, and pull in. We also studied activities of Na + , K + -ATPase and Ca 2 + -activated Mg 2 + -dependent ATPase in the absence and in the presence MLO venom. Venom was added into the assay mixture with low, sub-lethal (0.35 mg/kg approx. 0.5 LD 50 for rat) and lethal Abstracts e109
doi:10.1016/j.bbabio.2014.05.262 fatcat:5rym3hoz5rbpzkynihy45ztpia