Voltage-Dependent Interaction of Capsaicine and Protons on TRPV1-Receptors.

The interaction of TRPV1-receptors agonists (capsaicin and protons) has been studied on cultured CHO cells transfected by TRPV1-receptors. Using the whole-cell patch-clamp approach, it was shown that summation of the currents induced by agonist application was dependent on the membrane potential. The TRPV1-mediated currents induced by the pH and Capsaicin demonstrated arithmetical summation at potentials between 40--40 mV, while they were potentiated at potentials below -40 mV. Currents induced by the pH and Capsaicin combined were higher in comparison with the arithmetic sum of the currents induced by the pH and Capsaicin applied separately at such potentials. Such a potential dependence seems to be a base of the sensitization that is induced by inflammation or pain, when concentrations of proinflammatory mediators acting as TRPV1 agonists are increasing. Further depolarization induced by TRPV1 activation doesn't generate potentiation, which might serve as a protective mechanism to restrict their activity.

Design of antagonists for NMDA and AMPA receptors.

Determinants of antagonism of NMDA and calcium permeable AMPA receptor channels by organic cations were studied using several homologous series of mono- and dicationic derivatives of adamantane, phenylcyclohexyl, triphenylmethane, diphenylmethane. Antagonism by these drugs was studied on native receptors of isolated rat brain neurons and on recombinant GluR1 receptors expressed by Xenopus oocytes. The major action of these compounds was on the open channel, although minor competitive or closed channel antagonism cannot be ruled out. Analysis of structure-activity relationships suggests that all organic monocations are selective antagonists of NMDA receptors. Compounds exhibiting trapping block are more potent than those exhibiting weakly-trapping block. AMPA and NMDA receptor channels are blocked by dicationic organic compounds, the former requiring a certain distance between the hydrophobic moiety and the terminal charged group. Variations of their terminal ammonium group demonstrated that trimethylammonium derivatives are the most potent antagonists of AMPA receptors, whereas the terminal amino group is optimal for block of NMDA receptors. Based on the action of 38 compounds, topographical models of the binding sites of these compounds on NMDA and AMPA receptor channels are presented. These models will help to design channel-blocking drugs with defined potency and selectivity of action.

Determinants of trapping block of N-methyl-d-aspartate receptor channels.

Open channel blockers of NMDA receptors interact with the channel gate in different ways. Compounds like MK-801 and phencyclidine exhibit pronounced trapping block, whereas 9-aminoacridine and tetrapentylammonium cannot be trapped. Some blockers such as memantine and amantadine exhibit intermediate properties, so called 'partial trapping'. To analyze the determinants of trapping we have synthesized a series of mono- and dicationic derivatives of phenylcyclohexyl. The blocking action of these compounds as well as that of amantadine has been studied on native NMDA receptors of hippocampal pyramidal neurons. Use-dependence and kinetics of the blockade have been analyzed to estimate the degree of trapping. Dimensions of the blocking molecules apparently do not correlate with their trapping. However, the degree of trapping is voltage-dependent and correlates with the kinetics of unblock. For instance, amantadine behaved as non-trapping blocker at positive voltages, but demonstrated significant trapping at negative voltages. The data may be explained by the model in which the NMDA receptor channel has two binding sites: the shallow and deep ones. Binding to the deep but not to the shallow site allows trapping of the blockers.

Characterization of acid-sensitive ion channels in freshly isolated rat brain neurons.

Transient proton-activated currents induced by rapid shifts of the extracellular pH from 7.4 to < or =6.8 were recorded in different neurons freshly isolated from rat brain (hypoglossal motoneurons, cerebellar Purkinje cells, striatal giant cholinergic interneurons, hippocampal interneurons, CA1 pyramidal neurons and cortical pyramidal neurons) using whole-cell patch clamp technique. Responses of hippocampal CA1 pyramidal neurons were weak (100-300 pA) in contrast to other types of neurons (1-3 nA). Sensitivity of neurons to rapid acidification varied from pH(50) 6.4 in hypoglossal motoneurons to 4.9 in hippocampal interneurons. Proton-activated currents were blocked by amiloride (IC(50) varied from 3.6 to 9.5 microM). Reversal potential of the currents was close to E(Na), indicating that the currents are carried by sodium ions. The data obtained suggest that the proton-activated currents in the neurons studied are mediated by acid-sensitive ion channels. Strong acidification (pH<4) induced biphasic responses in all neuron types: the transient current was followed by a pronounced sustained one. Sustained current was not blocked by amiloride and exhibited low selectivity for sodium and cesium ions. Slow acidification from pH 7.4 to 6.5 did not induce detectable whole-cell currents. At pH 6.5, most of the channels are desensitized and responses to fast pH shifts from this initial level are decreased at least 10 times. This suggests that slow acidification which is well known to accompany some pathological states should rather desensitize than activate acid-sensitive ion channels and depress their function. Our results provide evidence for a widespread and neuron-specific distribution of acid-sensitive ion channels in the brain. The large amplitudes and transient character of currents mediated by these channels suggest that they could contribute to fast neuronal signaling processes.

Pharmacological analysis of the subunit composition of the AMPA receptor in hippocampal neurons.

Experiments were performed on isolated neurons from hippocampal field CA1 and the dentate fascia to identify the subunit composition and distribution of splicing variants of AMPA receptor subunits. Currents evoked by the application of kainate were recorded using a whole-cell patch clamping method. The presence of GluR2 subunits in receptors was associated with a sharp reduction in the activity of the selective channel blocker IEM-1460. The composition of flip versions of subunits was assessed using cyclothiazide. AMPA receptors in the major cell types (pyramidal and granule cells) had low sensitivity to IEM-1460, while AMPA receptors of other cells (interneurons) had high or intermediate sensitivity. Cyclothiazide had strong potentiating effects on the main cell types in both structures as compared with interneurons. Thus, there is a correlation between the sensitivities of hippocampal neurons to IEM-1460 and cyclothiazide. The main cell types in both structures expressed large quantities of the GluR2 subunit in their AMPA receptors, with high levels of flip subunits, as compared with the other cell types, in which GluR2 subunits were virtually absent and the flop version predominated. This appears to reflect the functional features of different types of neurons.

Ca2+-dependent desensitization of AMPA receptors.

The effect of changes in the external concentrations (0.4-10 mM) of Ca2+ ions on AMPA receptors (AMPARs) of different subunit composition was studied on freshly isolated rat brain neurones. Ca2+ produces rapid and reversible voltage-independent inhibition of AMPARs. Ca2+-permeable and Ca2+-impermeable AMPARs are equally sensitive to external Ca2+ suggesting that the effect is not addressed to the ion channel. The inhibition of responses evoked by AMPA is significantly larger than those evoked by kainate or glutamate. Cyclothiazide and aniracetam, which are known to prevent AMPAR desensitization, both greatly diminish inhibition of AMPARs by Ca2+. Cyclothiazide is more potent than aniracetam in both preventing of AMPAR desensitization and protecting against the Ca2+ inhibitory effect on hippocampal pyramidal cells. On giant cholinergic interneurones of striatum, aniracetam but not cyclothiazide significantly prevents inhibition by Ca2+. This agrees with available data on relative abundance of flip and flop splice variants in these cell types. The results suggest that Ca2+ may allosterically increase AMPA receptor desensitization independently on subunit composition and splice variants.

Different arrangement of hydrophobic and nucleophilic components of channel binding sites in N-methyl-D-aspartate and AMPA receptors of rat brain is revealed by channel blockade.

In order to investigate the topography of the channel binding site in (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) types of glutamate receptors, we have compared the blocking actions of mono- and dicationic derivatives of adamantane and phenylcyclohexyl. The potencies of dicationic derivatives to block AMPA receptor channels are about 1000 times higher than those of monocationic ones, whereas NMDA receptors are equally sensitive to both mono- and dicationic derivatives. The dependence of the activity of dicationic compounds on the length of the polymethylene chain between ammonium groups has a pronounced maximum for AMPA receptor channel block. For NMDA receptor channel dicationic compounds with various internitrogen distances produce similar blocking effects. The results show that hydrophobic and nucleophilic components of the binding site are located close to each other in the NMDA receptor channel but are separated by approximately 10 A in the AMPA receptor channel.