Ion-permeability properties of ionotropic glutamate receptors and their use as detectors in capillary electrophoresis

The aims of the thesis were to characterize ion-permeability properties of AMPA receptors expressed in interneurons from rat olfactory bulb and Xenopus oocytes injected by total RNA from human epileptic temporal lobe and, to develop a method using patch clamped cells as detector systems in capillary electrophoresis. The Ca2+-permeability of AMPA receptors was studied in interneurons from rat olfactory bulb. The interneurons were either acutely isolated or from primary cultures. The ion-permeability were studied by means of patch clamp. Kainate (KA) was used to activate AMPA mediated currents. The data from current-to-voltage relationships were fitted with the constant field equation or the Eyring rate theory. Functional AMPA receptors were expressed in Xenopus oocytes by injection of total RNA from human epileptic temporal lobe.The expressed AMPA receptors, activated by KA, were studied with two-electrode voltage clamp, in order to evaluate their Ca2+-permeability properties. Interneurons from the rat olfactory bulb were used as the detectors in a capillary elctrophoresis- patch clamp (CE-PC) analysis of biomolecules that activate or inhibit ligand-gated ion-channels. In CE-PC detection of antagonists, it is necessary to deliver constantly an agonist to the cell surface of the detector. This was performed by adding the specific agonist to the electrolyte solution contained in the electrophoresis capillary. In interneurons from the olfactory bulb, the current responses to kainate showed a linear/outwardly rectifying current-to-voltage relationship. Increasing the Ca2+ concentration from 1 mM to 60 mM gave a leftward the average reversal potential from +7 mV to -32 mV. The permeability properties of the AMPA receptors expressed by interneurons from rat olfactory bulb were well predicted by the Eyring rate theory. The model gave a pCa2+/pK+- permeability ratio of 0.06 for acutely isolate interneurons and 0.14 for interneurons in primary culture. The constant field equation gave corresponding low permeability ratios of 0.18 and 0.40 for acutely isolated cells and cells in primary culture, respectively. Thus it was concluded that interneurons in the olfactory bulb mainly express AMPA receptors with low permeability to Ca2+. Administration of kainate to Xenopus oocytes injected with total RNA from human epileptic temporal lobe gave non-desensitizing AMPA receptor mediated currents. The current-to-voltage relationhip showed an inward rectification and when the concentration of divalent cations were increased, there was a shift in the reversal potential from -11 mV (2mM Ca2+) to 12 mV (60 mM Ba2+). This yielded a pBa2+/pK+ permeability ratio of 1.6 when the constant field equation was used. It was concluded that the AMPA receptors from the human epileptic temporal lobe had a pronounced permeability to Ca2+. Mixtures of *-aminobutyrate, L-glutamate, N-methyl-D-aspartate (NMDA) and kainate (KA) were separated and detected by CE-PC. Also a mixture of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dichloro-3-hydroxy-2-quinoxaline-carboxylate (DiCl-HQC) were separated and detected by KA activated patch-clamp interneuron from rat olfactory bulb. Mg2+ that reversibly blocks the NMDA receptor in a voltage dependent way, was detected by the same detection system when activated by electrophoretically delivered NMDA and the co-agonist glycine. We have developed a CE-PC detection system that uses cell membranes from cells. The CE-PC system allows us to identify biologically active analytes in complex mixtures or samples. A broad spectrum of information including response characteristics, current-to-voltage relationships, dose-response measurements, kinetics and ion-channel conductance levels were extracted from the recordeD currents evoked by electrophoretically separated receptor agonists.The present method also offers new possibilities for drug screening and for identifying endogenous receptor antagonists and to determine their mode of action on any ionotropic receptor of interest

Probing Structure and Function of Ion Channels Using Limited Proteolysis and Microfluidics

Even though gain, loss, or modulation of ion channel function is implicated in many diseases, both rare and common, the development of new pharmaceuticals targeting this class has been disappointing, where it has been a major problem to obtain correlated structural and functional information. Here, we present a microfluidic method in which the ion channel TRPV1, contained in proteoliposomes or in excised patches, was exposed to limited trypsin proteolysis. Cleaved-off peptides were identified by MS, and electrophysiological properties were recorded by patch clamp. Thus, the structure-function relationship was evaluated by correlating changes in function with removal of structural elements. Using this approach, we pinpointed regions of TRPV1 that affect channel properties upon their removal, causing changes in current amplitude, single-channel conductance, and EC50 value toward its agonist, capsaicin. We have provided a fast "shotgun" method for chemical truncation of a membrane protein, which allows for functional assessments of various peptide regions

Biphasic modulation of NMDA-induced responses in pyramidal cells of the medial prefrontal cortex by Y-931, a potential atypical antipsychotic drug

Similar to the effects produced by the atypical antipsychotic drugs (APDs) clozapine and olanzapine, Y-931 {8-fluoro-12(4-methylpiperazin-1-yl)-6H-[1]benzothieno[2,3-b][1,5] benzodiazepine maleate, a purported atypical APD} effectively facilitated N-methyl-D-aspartate (NMDA)-induced, but not (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-evoked, responses in pyramidal cells of the rat medial prefrontal cortex (mPFC). Similar to olanzapine and clozapine, the concentration-response curve of Y-931 in these experiments was biphasic. At present, the mechanisms behind the biphasic modulatory actions of Y-931 and olanzapine on NMDA-induced currents in the mPFC are not clear. In addition to augmenting NMDA responses, Y-931 prevented the phencyclidine (PCP)-induced block of the NMDA responses and increased the amplitudes and durations of excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation of the forceps minor. Overall, our findings suggest that APDs, particularly the atypical ones, share a common property in that they facilitate NMDA receptor-mediated transmission in the mPFC and perhaps other functionally related limbic structures as well, which could be the cellular basis for their ability to alleviate some schizophrenic negative symptoms and cognitive dysfunctions. © 2001 Wiley-Liss, Inc

A Heating-Superfusion Platform Technology for the Investigation of Protein Function in Single Cells

Here, we report on a novel approach for the study of single-cell intracellular enzyme activity at various temperatures, utilizing a localized laser heating probe in combination with a freely positionable microfluidic perfusion device. Through directed exposure of individual cells to the pore-forming agent a-hemolysin, we have controlled the membrane permeability, enabling targeted delivery of the substrate. Mildly permeabilized cells were exposed to fluorogenic substrates to monitor the activity of intracellular enzymes, while adjusting the local temperature surrounding the target cells, using an infrared laser heating system. We generated quantitative estimates for the intracellular alkaline phosphatase activity at five different temperatures in different cell lines, constructing temperature-response curves of enzymatic activity at the single-cell level. Enzymatic activity was determined rapidly after cell permeation, generating five-point temperature-response curves within just 200 s

A Heating-Superfusion Platform Technology for the Investigation of Protein Function in Single Cells

Here, we report on a novel approach for the study of single-cell intracellular enzyme activity at various temperatures, utilizing a localized laser heating probe in combination with a freely positionable microfluidic perfusion device. Through directed exposure of individual cells to the pore-forming agent a-hemolysin, we have controlled the membrane permeability, enabling targeted delivery of the substrate. Mildly permeabilized cells were exposed to fluorogenic substrates to monitor the activity of intracellular enzymes, while adjusting the local temperature surrounding the target cells, using an infrared laser heating system. We generated quantitative estimates for the intracellular alkaline phosphatase activity at five different temperatures in different cell lines, constructing temperature-response curves of enzymatic activity at the single-cell level. Enzymatic activity was determined rapidly after cell permeation, generating five-point temperature-response curves within just 200 s

Bicarbonate-sensitive cysteine induced elevation of extracellular aspartate and glutamate in rat hippocampus in vitro

The effect of different concentrations of cysteine (0.125, 0.25, 0.5 and 1 mM) on the net efflux of endogenous amino acids was studied by the incubation of rat hippocampal slices. Addition of cysteine (1 mM) in bicarbonate containing low K+ medium (5 min) selectively increased the basal net efflux of glutamate and aspartate by 370% and 396%, respectively. High K+ media (50 mM) containing cysteine (1 mM) evoked the net efflux of glutamate and aspartate by 1 454% and 1 019%, respectively. The corresponding effects in control slices without cysteine were 669% and 404%, respectively. No changes were observed on the concentrations of GABA, glutamine and taurine. The cysteine oxidation products, cysteine sulfinate (0.5 μM) and cystine (0.25 mM) were without effects. The effect of cysteine (0.5 mM) was dramatically reduced in media with no added bicarbonate/CO2. Thus, cysteine in a bicarbonate-sensitive manner selectively increases the extracellular concentration of excitotoxic amino acids in adult rat brain in vitro, possibly by interfering with the carrier-mediated glutamate uptake/ releas