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The kinetics of open channel block of GluR2‐containing and GluR2‐lacking AMPA receptors (AMPAR) by dicationic compounds (IEM‐1460, IEM‐1754, and IEM‐1925) have been studied in rat hippocampal neurones using whole‐cell patch clamp recording and concentration‐jump techniques. Neurones were isolated from hippocampal slices by vibrodissociation.The dicationic compounds were approximately 100–200 times more potent as blockers of GluR2‐lacking AMPAR than as blockers of GluR2‐containing AMPAR. The subunit specificity of channel block is determined by the blocking rate constant of a dicationic compound, whereas differences in unblocking rate constants account for differences in potency.Hyperpolarization may decrease the block produced by IEM‐1460 and IEM‐1754 block due to the voltage‐dependence of the unblocking rate constants for these compounds. This suggests that dicationic compounds permeate the AMPAR channel at negative membrane potentials. The effect was particularly apparent for GluR2‐lacking AMPAR. These findings indicate that the presence of GluR2‐subunit(s) in AMPAR hinders the binding of the cationic compounds and their permeation through the channel.The most potent compound tested was IEM‐1925. The presence of a phenylcyclohexyl moiety instead of an adamantane moiety, as in IEM‐1460 and IEM1754, is probably responsible for the higher potency of IEM‐1925. Dicationic compounds are important not only as pharmacological tools, but also as templates for the synthesis of new selective AMPAR blockers which may be potential therapeutic agents.

We examined the influence of the molecular structure of four novel adamantane derivatives on their ability to block the channels of nicotinic acetylcholine (ACh) and N-methyl-D-aspartate (NMDA) receptors. The structure of the drugs is Ad-CH2-N+H2-(CH2)5-R, where Ad is adamantane and R was varied from ammonium (IEM-1754) to tert-butyldimethylammonium (IEM-1857) radical. The compounds induced double-exponential decays of postsynaptic currents in frog muscles and flickering of NMDA-activated channels, suggesting that each drug acts as a fast open-channel blocker at both types of receptors. The equilibrium dissociation constants (Kd) of the drugs for ACh-activated channels at -80 mV were similar, whereas the Kd values for NMDA-activated channels at -80 mV were 2-10 times lower. Several observations suggested that occupation of either type of channel by these compounds inhibited channel closure; the time constant (tau) of the slow component of the decay of postsynaptic currents in the presence of each compound was greater than the control tau, the IC50 of IEM-1754 for inhibition of NMDA-activated whole-cell currents was > 20 times larger than its Kd for the open channel, and a transient increase in NMDA-activated whole-cell currents was observed after washout of IEM-1754. Thus, these drugs appear to act on nicotinic ACh and NMDA receptors via similar mechanisms, although the voltage dependence of block suggested that the drugs bind at a more superficial site in the ACh-activated channel. All compounds also potently prevented NMDA-induced convulsions in mice. The ED50 of IEM-1754 was 4 times lower than the ED50 of MK-801.

The blocking effects of newly synthesized compounds N-decyltropine bromide (IEM-1556) and its derivatives N-methyltropine iodide (IEM-1893) and N-hexyltropine iodide (IEM-1848), possessing aliphatic radicals of different length, on the vagus-induced reduction of the heart rate were studied in acute experiments on rats. The effects produced by these compounds on the level of arterial blood pressure and synaptic transmission in the superior cervical ganglion (SCG) of the rat were studied and compared with those produced by classical ganglion-blocking agents (hexamethonium and trimetaphan) and muscle relaxants (pancuronium and decamethonium). IEM-1556 much more effectively blocked the vagus-induced reduction of the heart rate than other tested blocking agents, and, in contrast to the classical ganglion-blocking agents and decamethonium, did not reduce the arterial blood pressure. In addition, IEM-1556 was less effective than hexamethonium in blocking synaptic transmission through the SCG. IEM-1893 and IEM-1848 demonstrated lower efficacy in blocking SCG transmission, if compared with that of IEM-1556. The results suggest that IEM-1556 is a highly selective blocking agent for parasympathetic versus sympathetic pathways, and its selectivity is determined by the presence of a decyl aliphatic radical in its molecule.

The effects of d-sparteine (d-SP) and its two derivatives, N-methylsparteine (IEM-1820) and N-phenylsparteine (IEM-1821), on nicotinic acetylcholine receptors (nAChR) of the rat superior cervical ganglion neurons were studied. Membrane currents evoked by iontophoretically applied acetylcholine were recorded using the patch-clamp recording technique in the whole-cell configuration. All three compounds were found to block nAChR competitively, the blocking activity being increased with an increase in the size of the blocking molecule. The EC50 values for d-SP, IEM-1820, and IEM-1821 were equal to 2.06±0.38 µM (n=3), 1.64±0.41 µM (n=4), and 0.65±0.17 µM (n=3), respectively. It was assumed that the increase in efficiency of blocking is related to the decrease in the rate of dissociation of the blocker and receptor molecules.

The action of polymethylene bis-ammonium compounds on acetylcholine-activated channels was investigated on voltage-clamped neurons of the isolated rabbit superior cervical ganglion. The kinetics of complex formation by the compound and the open channel was determined from shortening of decay of the fast excitatory postsynaptic current, the rate of which corresponds to the rate of channel closure, whereas the kinetics of dissociation of this complex was determined by recovery of the second response to application of two pulses of acetylcholine. With membrane hyperpolarization complex formation was found to be accelerated, and its dissociation retarded. The potential-dependence of interaction between compound and channel increases with lengthening of the polymethylene chain. Variation of one of the two cationic groups in a compound with a tetramethylene chain does not affect potential-dependence. In a series of bis-ammonium compounds, ganglion-blocking activity, determined on the cat superior cervical ganglionin situ was found to correlate with the velocity constant of binding with the open channel. It is concluded that the ganglion-blocking action of bis-ammonium compounds is determined by their channel-blocking activity.

1The effects of two adamantane derivatives, 1‐trimethylammonio‐5‐(l‐adamantane‐methylammoniopentane dibromide) (IEM‐1460) and 1‐ammonio‐5‐(l‐adamantane‐methylammoniopentane dibromide) (IEM‐1754) on kainate‐induced currents were studied in Xenopusoocytes expressing recombinant ionotropic glutamate receptors and in freshly isolated neurones from rat hippocampal slices.2The adamantane derivatives caused use‐and voltage‐dependent block of open channels of recombinant AMPA receptors. This antagonism was dependent on receptor subunit composition; channels gated by recombinant, homomeric GluRl and GluR3 receptors exhibited a higher sensitivity to block than those gated by receptors containing edited GluR2 subunits. In the former cases, IEM‐1460 had an IC50of 1.6 μmat a holding potential (Vh) of −80 mV and IEM‐1754 was 3.8 times less potent than IEM‐1460. In contrast, 100 μmIEM‐1460 inhibited responses to 100 μmkainate of receptors containing edited GluR2 subunits by only 7.8 ± 2.4% (n= 5oocytes) at a Vhof −80 mV.3Native AMPA/kainate receptors in isolated hippocampal cells were inhibited by adamantane derivatives in a use‐ and voltage‐dependent manner. This antagonism was dependent on cell type: pyramidal neurones were less sensitive to IEM‐1460 (IC50= 1617 μmat Vh=−80mV) than interneurones (IC50= 1.6 μmat Vh=−80 mV). IEM‐1460 and IEM‐1754 were equipotent when applied to pyramidal neurones, but IEM‐1754 was less potent (∼3 times) than IEM‐1460 when applied to interneurones.4It is concluded that the presence of the edited GluR2 subunit in recombinant AMPA receptors and native AMPA/kainate receptors inhibits channel block by organic cations and that adamantane derivatives are potentially valuable tools for identifying classes of AMPA/kainate receptors and their roles in synaptic transmission.

The action of three dicationic drugs, derivatives of adamantane (IEM-1460 and IEM-1754) and phencyclidine (IEM-1925), on glutamate receptors (GluRs) at the insect neuromuscular junction (Calliphora vicinalarva) and on neurons of the freshwater gastropodian molluscPlanorbarius corneushas been studied using the voltage clamp technique. In the presence of concanavalin A complex glutamate-induced currents recorded from molluscan neurons reflected mainly the opening of cationic channels as a result of decreased desensitisation and inhibition of a chloride component. Under these conditions all drugs studied inhibited the stationary component of glutamate-gated cationic currents in a dose-dependent manner (IC50s were 0.1 μM, 1.0 μM and 19.2 μM for the action of IEM-1925, IEM-1460 and IEM-1754 respectively). The same rank order of potency: IEM-1925 > IEM-1460 > IEM-1754 was observed in both the insect and mollusc. The results of these experiments are compared with those obtained earlier on vertebrate GluRs. Open-channel blocking drugs may help to identify and classify GluRs of invertebrates, and could be used as tools to elucidate the involvement of GluRs in the transmission at certain synapses.