A vast body of experimental in vitro work and modelling studies suggests that the firing pattern and/or rate of a majority of midbrain dopaminergic neurons may be controlled in part by Ca2+-activated K+ channels of the SK type. However, due to the lack of suitable tools, in vivo evidence is lacking. We have taken advantage of the development of the water-soluble, medium potency SK blocker N-methyl-laudanosine (CH3-L) to test this hypothesis in anaesthetized rats. In the lateral ventral tegmental area, CH3-L iontophoresis onto dopaminergic neurons significantly increased the coefficient of variation of their interspike intervals and the percentage of spikes generated in bursts as compared to the control condition. The effect of CH3-L persisted in the presence of a specific GABA(A) antagonist, suggesting a direct effect. It was robust and reversible, and was also observed in the substantia nigra. Control experiments demonstrated that the effect of CH3-L could be entirely ascribed to its blockade of SK channels. On the other hand, the firing pattern of noradrenergic neurons was much less affected by CH3-L. We provide here the first demonstration of a major role of SK channels in the control of the switch between tonic and burst firing of dopaminergic neurons in physiological conditions. This study also suggests a new strategy to develop modulators of the dopaminergic (DA) system, which could be of interest in the treatment of Parkinson's disease, and perhaps other diseases in which DA pathways are dysfunctional.

Small conductance calcium-activated potassium (SK) channels are found in many types of neurons as well as in some other cell types. These channels are selective for K(+) and open when intracellular Ca(2+) rises to omega 500 nM. In neurons, this occurs during and after an action potential. Activation of SK channels hyperpolarizes the membrane, thus reducing cell excitability for several tens or hundreds of milliseconds. This phenomenon is called a afterhyperpolarization (AHP). Three subtypes of SK channels (SK1, SK2, SK3) have been cloned and exhibit a differential localization in the brain. SK channels may play a role in physiological and pathological conditions. They may be involved in the control of memory and cognition. Moreover, they are heavily expressed in the basal ganglia (in particular in the substantia nigra, pars compacta) and in the limbic system, suggesting that they may modulate motricity and emotional behaviour. Based on these facts, SK channel subtypes may be a suitable target for developing novel therapeutic agents, but more work is needed to validate these targets. Hence, there is a great need for selective ligands. Moreover, although the risk of peripheral side-effects for SK channel modulators appears to be low, some questions remain to be investigated. Currently, different molecules are known as SK channel modulators. Apamin is a very potent peptidic agent; it produces a strong blockade of these targets which is only very slowly reversible and it has limited selectivity. Dequalinium was found to be an effective blocker. Different chemical modulations on the dequalinium structure led to the discovery of highly potent bis-quinolinium derivatives such as UCL 1684. Other bis-(2-amino-benzimidazole) derivatives are in development. On the other hand, quaternary salts of bicuculline were reported to be effective in inhibiting AHPs. More recent developments on structurally-related molecules revealed that methyl-laudanosine is a new interesting tool for exploring SK channel pharmacology. Finally, a family of compounds has been shown to facilitate SK channel opening. Such compounds may be useful in treating disorders involving neuronal hyperexcitability.

1 We have recently shown that the alkaloid methyl-laudanosine blocks SK channel-mediated afterhyperpolarizations (AHPs) in midbrain dopaminergic neurones. However, the relative potency of the compound on the SK channel subtypes and its ability to block AHPs of other neurones were unknown. 2 Using whole-cell patch-clamp experiments in transfected cell lines, we found that the compound blocks SK1, SK2 and SK3 currents with equal potency: its mean IC 50 s were 1.2, 0.8 and 1.8 mM, respectively. IK currents were unaffected. In rat brain slices, methyl-laudanosine blocked apaminsensitive AHPs in serotonergic neurones of the dorsal raphe and noradrenergic neurones of the locus coeruleus with IC 50 s of 21 and 19 mM, as compared to 15 mM in dopaminergic neurones. However, at 100 mM, methyl-laudanosine elicited a constant hyperpolarization of serotonergic neurones of about 9 mV, which was inconsistently (i.e. not in a reproducible manner) antagonized by atropine and hence partly due to the activation of muscarinic receptors. 3 While exploring the pharmacology of related compounds, we found that methyl-noscapine also blocked SK channels. In cell lines, methyl-noscapine blocked SK1, SK2 and SK3 currents with mean IC 50 s of 5.9, 5.6 and 3.9 mM, respectively. It also did not block IK currents. Methyl-noscapine was slightly less potent than methyl-laudanosine in blocking AHPs in brain slices, its IC 50 s being 42, 37 and 29 mM in dopaminergic, serotonergic and noradrenergic neurones, respectively. Interestingly, no significant non-SK effects were observed with methyl-noscapine in slices. At a concentration of 300 mM, methyl-noscapine elicited the same changes in excitability in the three neuronal types than did a supramaximal concentration of apamin (300 nM). 4 Methyl-laudanosine and methyl-noscapine produced a rapidly reversible blockade of SK channels as compared with apamin. The difference between the IC 50 s of apamin (0.45 nM) and methyllaudanosine (1.8 mM) in SK3 cells was essentially due to a major difference in their k À1 (0.028 s À1 for apamin and X20 s À1 for methyl-laudanosine). 5 These experiments demonstrate that both methyl-laudanosine and methyl-noscapine are medium potency, quickly dissociating, SK channel blockers with a similar potency on the three SK subtypes. Methyl-noscapine may be superior in terms of specificity for the SK channels.

Small conductance calcium-activated potassium channels (SK) are widely expressed throughout the central nervous system (CNS) and the periphery. Three subtypes of SK channels have so far been identified in different parts of the brain. Activation of the SK channels by a rise in intracellular calcium leads to the hyperpolarisation of the membrane, reducing cell excitability. Blocking the SK channels might be beneficial in the treatment of depression, Parkinson's disease and cognitive disorders. However, few blockers of SK channels have been characterized. In this study, a pharmacophoric model of SK channels blockers is presented. It is based on a series of nonpeptidic compounds and apamin, a peptidic blocker. To create the pharmacophore model, the conformational space of nonpeptidic blockers was investigated to generate a series of distance constraints applied to a simulated annealing study of apamin. The resulting conformation was superimposed with the nonpeptidic blockers to give a pharmacophore.