Activation of small conductance calcium-activated potassium (K Ca 2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin apamin, but the mechanism of block is not understood. For example, apamin binds to both K Ca 2.2 and K Ca 2.3 with the same high affinity (K D ϳ 5 pM for both subtypes) but requires significantly higher concentrations to block functional current (IC 50 values of ϳ100 pM and ϳ5 nM, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that apamin blocks K Ca 2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.K Ca 2 channels (formerly known as SK channels) are characterized by their sensitivity to the highly specific toxin apamin (1). This 18-amino acid peptide, which has been isolated from the honeybee (Apis mellifera) venom (2), contains two disulfide bridges that provide a fairly rigid tertiary conformation (3), with two arginine residues (Arg-13 and Arg-14) being critical for its biological activity (4). The cloning of K Ca 2 channel subunits has revealed the existence of three subtypes (K Ca 2.1-K Ca 2.3, formerly SK1-SK3) (5) that bind apamin with very high affinity (K D ϳ 5-10 pM) (see Ref. 6 for a review). However, apamin is less potent at blocking K Ca 2 current and displays differential block of channel subtypes. For example, K Ca 2.2 (all species) displays the highest sensitivity, with IC 50 values from 27 to 140 pM. Rat, human, and mouse K Ca 2.3-mediated currents show an intermediate sensitivity, with IC 50 values ranging from 0.63 to 19 nM. Finally, human K Ca 2.1 is the least sensitive, with reported IC 50 values ranging between 0.7 and 100 nM (6). These differences between binding and electrophysiological results suggest that the mechanism of block by apamin is complex and that binding and block by the toxin are not identical phenomena.K Ca 2 channel subtypes are expressed throughout the CNS and periphery, displaying partially overlapping but distinct locations. This has led to the proposal that block of K Ca 2 channels may be a novel t...

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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.

Activation of small-conductance calcium (Ca 2+ )-dependent potassium (K Ca 2) channels (herein called "SK") produces membrane hyperpolarization to regulate membrane excitability. Three subtypes (SK1-3) have been cloned and are distributed throughout the nervous system, smooth muscle, and heart. It is difficult to discern the physiological role of individual channel subtypes as most blockers or enhancers do not discriminate between subtypes. The archetypical blocker apamin displays some selectivity between SK channel subtypes, with SK2 being the most sensitive, followed by SK3 and then SK1. Sensitivity of SK1 is species specific, with the human isoform being blocked by the toxin, whereas the rat is not. Mutation studies have identified residues within the outer pore that suggest apamin blocks by an allosteric mechanism. Apamin also uses a residue within the S3-S4 extracellular loop to produce a high-sensitivity block. We have identified that a 3-amino acid motif within this loop regulates the shape of the channel pore. This motif is required for binding and block by apamin, suggesting that a change in pore shape underlies allosteric block. This motif is absent in rat SK1, explaining why it is insensitive to block by apamin. The overlapping distribution of SK channel subtype expression suggests that native heteromeric channels may be common. We show that the S3-S4 loop of one subunit overlaps the outer pore of the adjacent subunit, with apamin interacting with both regions. This arrangement provides a unique binding site for each combination of SK subunits within a coassembled channel that may be targeted to produce blockers specific for heteromeric SK channels.patch clamp | afterhyperpolarization P harmacological modulation of small-conductance Ca 2+ -activated potassium (SK/K Ca 2) channels has been suggested as a potential target for a number of disorders including dementia, depression, and cardiac arrhythmias. Three subtypes of SK channel have been cloned, with distinct but partially overlapping expression patterns (1-3). A nonpeptidic subtype-specific blocker of SK channels has yet to be developed. The lack of SK subtype selective blockers combined with this wide distribution means that their roles cannot be well defined. This lack of specificity also means that current available SK blockers have a very narrow therapeutic window, with an overlap of doses required for therapeutic benefit and those producing toxic effects in animal studies (4). SK channels are blocked by the bee venom toxin apamin, which displays weak subtype selectivity. The toxin is most potent at SK2 (IC 50 ∼ 70 pM) (5-9) and SK3 (IC 50 ∼ 0.63-6 nM) (7, 9, 10), followed by the human isoform of SK1 (IC 50 ∼ 1-8 nM) (8, 9), whereas the rat isoform of SK1 is apamin insensitive (11,12). Mutation studies have identified a number of residues within the outer pore that affect block by apamin (7,8,13). For example, an outer pore histidine residue has been shown to be critical for both binding and block by the toxin (7). Structural modeling has place...

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.