Researchers at the Department of Neurochemistry are involved in projects related to mechanisms underlying diseases characterized by metabolic deficits and synaptic impairment such as epilepsy and ischemia. Biochemical, electrophysiological, imaging and molecular modeling techniques are used to disclose basic mechanisms of neurotransmission as well as to explore pharmacologically relevant properties of potential drug targets.

Current research projects are to

 

Transporter-mediated coupling of Glu and GABA signaling

The main goal of this project is to explore the role of Glu and GABA transporters in the neuron-glia communication. By the application of ramifying biological models at different levels of complexity in combination with different analytical as well as pharmacological and anatomical approaches, we disclosed several lines of evidence that substrate activation of Glu transporters increases ambient GABA level both in vitro and in vivo. This action appears to be Ca2+ ion-independent acting through glial GABA transporter reversal and can be eliminated by preventing Glu uptake. We ruled out Glu or GABA receptor-mediated mechanisms that might have accounted for the release of GABA.

We described a new mechanism underlying the phenomenon by which coordinated activation of Glu and GABA transporters determine the ratio of the ambient concentrations of major excitatory and inhibitory neurotransmitters.

Homology model of the GABA transporter

This project is addressed to develop high throughput screening of potentially neuroprotective GABA transporter type 1 (GAT1) inhibitors by docking GABA and substrate inhibitors into the homology model of human GABA transporter subtype 1. The homology model is being built based on the high resolution crystal structure of LeuTAa (PDB code: 2a65), a bacterial homologue of the GABA transporter family (left panel). Molecular mechanics calculations revealed distinguishable GABA docking to amino acid residues of TM1 & TM8 or TM1 & TM6 helices (highlighted in yellow or gray in the right panel, respectively).

Role of high-frequency field potential oscillations in ictogenesis

This project is aimed to shed light on mechanisms and neuronal network elements contributing to the synchronization that is a hallmark of epileptiform activity. Using electrophysiological approaches in combination with wavelet decomposition of the recorded signals we recently disclosed that the preictal discharges and the seizure-like events start by high-frequency field potential oscillations that in turn have a clear correlate in synaptic input recordings. This fast signal may contribute to ictogenesis and ictal synchronization.

The effect of succinate in the brain

A synaptic GHB recognition site, independent of GABAB receptor and interacting with the citric acid cycle intermediate succinate and a gap-junction inhibitor carbenoxolone (CBX), has recently been disclosed in the human nucleus accumbens (NA) and in the rat forebrain (Molnár et al., 2006; 2007). To address the presumed recognition site for succinate, we investigated the pharmacological profile of [3H]Succinate binding to synaptic membranes prepared from rat forebrain and human NA samples. We found an acidic pH-dependent, biphasic displacement profile of succinate distinguishing high- and low-affinity bindind sites.While GHB and lactate binded to both high- and low-affinity succinate binding sites, the gap-junction blockers CBX and flufenamic acid (FFA) together with the GHB binding site-selective NCS-382 ineracted only with the high-affinity binding site. Binding of the Na+/K+- ATPase inhibitor ouabain and citrate characterized the low-affinity succinate binding site. The half-maximal concentrations for inhibition (IC50) of different drugs are summarized in Figure 1.

Figure 1. Pharmacological characterization of [3H]succinate binding sites in synaptic membrane fractions of the rat forebrain. Data represents averages obtained from 2-8 determinations. Data are expressed as mean±SE. Displacement curves were fitted by one- or two-site competition approximation.

Currently we are working on a functional test for succinate in the brain, using the techniques of laser scanning confocal microscopy. We are investigating the effects of succinate compared to CBX on the ATP-generated intracellular Ca2+ ion signalling of nucleus accumbens cells. We plan to identify these cells and to explore possible relationships between succinate and functional gap-junctions.


Figure 2. The effect of carbenoxolone (CBX) and succinate on the fluorescent intensity of cells (loaded with the intracellular Ca2+-dye Fluo-4 AM) responding to ATP application. Data represent mean±SE from 4 (a) and 2 (b) rat nucleus accumbens slices.