The plant alkaloid methyllycaconitine (MLA) is considered to be a selective antagonist of the alpha7 subtype of neuronal nicotinic acetylcholine receptor (nAChR). However, 50 nM MLA partially inhibited (by 16%) [(3)H]dopamine release from rat striatal synaptosomes stimulated with 10 microM nicotine. Other alpha7-selective antagonists had no effect. Similarly, MLA (50 nM) inhibited [(3)H]dopamine release evoked by the partial agonist (2-chloro-5-pyridyl)-9-azabicyclo[4.2.1]non-2-ene (UB-165) (0.2 microM) by 37%. In both cases, inhibition by MLA was surmountable with higher agonist concentrations, indicative of a competitive interaction. At least two subtypes of presynaptic nAChR can modulate dopamine release in the striatum, and these nAChR are distinguished by their differential sensitivity to alpha-conotoxin-MII (alpha-CTx-MII). MLA was not additive with a maximally effective concentration of alpha-CTx-MII (100 nM) in inhibiting [(3)H]dopamine release elicited by 10 microM nicotine or 0.2 microM UB-165, suggesting that both toxins act at the same site. This was confirmed in quantitative binding assays with (125)I-alpha-CTx-MII, which displayed saturable specific binding to rat striatum and nucleus accumbens with B(max) values of 9.8 and 16.5 fmol/mg of protein, and K(d) values of 0.63 and 0.83 nM, respectively. MLA fully inhibited (125)I-alpha-CTx-MII binding to striatum and nucleus accumbens with a K(i) value of 33 nM, consistent with the potency observed in the functional assays. We speculate that MLA and alpha-CTx-MII interact with a presynaptic nAChR of subunit composition alpha3/alpha6beta2beta3* on dopamine neurons. The use of MLA as an alpha7-selective antagonist should be exercised with caution, especially in studies of nAChR in basal ganglia.
Muscarinic M1 acetylcholine receptors (M1Rs) are highly expressed in the hippocampus, and their inhibition or ablation disrupts the encoding of spatial memory. It has been hypothesized that the principal mechanism by which M1Rs influence spatial memory is by the regulation of hippocampal synaptic plasticity. Here, we use a combination of recently developed, well characterized, selective M1R agonists and M1R knock-out mice to define the roles of M1Rs in the regulation of hippocampal neuronal and synaptic function. We confirm that M1R activation increases input resistance and depolarizes hippocampal CA1 pyramidal neurons and show that this profoundly increases excitatory postsynaptic potential-spike coupling. Consistent with a critical role for M1Rs in synaptic plasticity, we now show that M1R activation produces a robust potentiation of glutamatergic synaptic transmission onto CA1 pyramidal neurons that has all the hallmarks of long-term potentiation (LTP): The potentiation requires NMDA receptor activity and bi-directionally occludes with synaptically induced LTP. Thus, we describe synergistic mechanisms by which acetylcholine acting through M1Rs excites CA1 pyramidal neurons and induces LTP, to profoundly increase activation of CA1 pyramidal neurons. These features are predicted to make a major contribution to the pro-cognitive effects of cholinergic transmission in rodents and humans.
Transient receptor potential vanilloid 3 (TRPV3) is a member of the TRP (Transient Receptor Potential) super-family. It is a relatively underexplored member of the thermo-TRP sub-family (Figure 1), however, genetic mutations and use of gene knock-outs and selective pharmacological tools are helping to provide insights into its role and therapeutic potential. TRPV3 is highly expressed in skin, where it is implicated in skin physiology and pathophysiology, thermo-sensing and nociception. Gain of function TRPV3 mutations in rodent and man have enabled the role of TRPV3 in skin health and disease to be particularly well defined. Pre-clinical studies provide some rationale to support development of TRPV3 antagonists for therapeutic application for the treatment of inflammatory skin conditions, itch and pain. However, to date, only one compound directed towards block of the TRPV3 receptor (GRC15300) has progressed into clinical trials. Currently, there are no known clinical trials in progress employing a TRPV3 antagonist.
Sazetidine-A has been recently proposed to be a "silent desensitizer" of α4β2 nicotinic acetylcholine receptors (nAChRs), implying that it desensitizes α4β2 nAChRs without first activating them. This unusual pharmacological property of sazetidine-A makes it, potentially, an excellent research tool to distinguish between the role of activation and desensitization of α4β2 nAChRs in mediating the central nervous system effects of nicotine itself, as well as those of new nicotinic drugs. We were surprised to find that sazetidine-A potently and efficaciously stimulated nAChR-mediated dopamine release from rat striatal slices, which is mediated by α4β2* and α6β2* subtypes of nAChR. The agonist effects on native striatal nAChRs prompted us to re-examine the effects of sazetidine-A on recombinant α4β2 nAChRs in more detail. We expressed the two alternative stoichiometries of α4β2 nAChR in Xenopus laevis oocytes and investigated the agonist properties of sazetidine-A on both α4(2)β2(3) and α4(3)β2(2) nAChRs. We found that sazetidine-A potently activated both stoichiometries of α4β2 nAChR: it was a full agonist on α4(2)β2(3) nAChRs, whereas it had an efficacy of only 6% on α4(3)β2(2) nAChRs. In contrast to what has been published before, we therefore conclude that sazetidine-A is an agonist of native and recombinant α4β2 nAChRs but shows differential efficacy on α4β2 nAChRs subtypes.
The realization of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one), described previously as a potent M1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that "pure" positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses.
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