Cédric Lamy

Allosteric Block of KCa2 Channels by Apamin

Activation of small conductance calcium-activated potassium (KCa2) 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 KCa2.2 and KCa2.3 with the same high affinity (KD ∼ 5 pm for both subtypes) but requires significantly higher concentrations to block functional current (IC50 values of ∼100 pm and ∼5 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 KCa2 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.

SK Channel blockade promotes burst firing in dorsal raphe serotonergic neurons

Previous in vivo studies have shown that blockade of small‐conductance Ca2+‐activated potassium (SK) channels enhances burst firing in dopaminergic neurons. As bursting has been found to be physiologically relevant for the synaptic release of serotonin (5‐HT), we investigated the possible role of SK channels in the control of this firing pattern in 5‐HT neurons of the dorsal raphe nucleus. In these cells, bursts are usually composed of doublets consisting of action potentials separated by a small interval (< 20 ms). Both in vivo and in vitro extracellular recordings were performed, using anesthetized rats and rat brain slices, respectively. In vivo, the specific SK blocker UCL 1684 (200 μm) iontophoresed onto presumed 5‐HT neurons significantly increased the production of bursts in 13 out of 25 cells. Furthermore, the effect of UCL 1684 persisted in the presence of both the GABAA antagonist SR 95531 (10 mm) and the GABAB antagonist CGP 35348 (10 mm), whereas these agents by themselves did not significantly influence the neuronal firing pattern. In vitro, bath superfusion of the SK channel blocker apamin (300 nm) induced bursting in only three out of 18 neurons, although it increased the coefficient of variation of the interspike intervals in all the other cells. Our results suggest that SK channel blockade promotes bursting activity in 5‐HT neurons via a direct action. An input which is present only in vivo seems to be important for the induction of this firing pattern in these cells.

Ion channel modulators: more diversity than previously thought

Ion‐channel function can be modified in various ways. For example, numerous studies have shown that currents through voltage‐gated ion channels are affected by pore block or modification of voltage dependence of activation/inactivation. Recent experiments performed on various ion channels show that allosteric modulation is an important mechanism for affecting channel function. For instance, in KCa2 (formerly SK) channels, the prototypic “blocker” apamin prevents conduction by an allosteric mechanism, while TRPV1 channels are prevented from closing by a tarantula toxin, DkTx, through an interaction with residues located away from the selectivity filter. The recent evidence, therefore, suggests that in several ion channels, the region around the outer mouth of the pore is rich in binding sites and could be exploited therapeutically. These discoveries also suggest that the pharmacological vocabulary should be adapted to define these various actions.

New pyridobenzoxazepine derivatives derived from 5-(4-methylpiperazin-1-yl)-8-chloro-pyrido[2,3-b][1,5]benzoxazepine (JL13): chemical synthesis and pharmacological evaluation

A series of new pyridobenzoxazepine derivatives with various heterocyclic amine side chains were synthesized to explore two main parameters related to the distal basic nitrogen. These compounds were tested for their affinity for dopamine D(2L) and D(4), serotonin 5-HT(1A) and 5-HT(2A), and adrenergic α(2A) receptors in comparison with 5-(4-methylpiperazin-1-yl)-8-chloro-pyrido[2,3-b][1,5]benzoxazepine, JL13 (1), and other diarylazepine derivatives. In terms of multireceptor target strategy, 2 and 5 present the most promising in vitro binding profile. Bulky, polar, and more flexible side chains are not favorable in this context. Compounds 2 and 5 were tested in adult rats to evaluate their long-term effects on dopamine and serotonin receptors density in different brain areas. Similar to 1 and other second-generation antipsychotic drugs, repeated treatment with 2 significantly increased D(1) and D(4) receptors in nucleus accumbens and caudate putamen and D(2) receptors in medial prefrontal cortex and hippocampus, while 5 significantly increased D(2) and D(4) receptors in nucleus accumbens. In addition, 2 increased 5-HT(1A) and decreased 5-HT(2A) receptors in cerebral cortex. In contrast, 5 did not alter levels of any 5-HT receptor subtype in any brain region examined. These results encourage further development of 2 as a novel second-generation antipsychotic agent.

Bis-tetrahydroisoquinoline derivatives: AG525E1, a new step in the search for non-quaternary non-peptidic small conductance Ca2+-activated K+ channel blockers

So far, small conductance Ca(2+)-activated K(+) channel (SK) blockers mostly consist of quaternary ammonium derivatives or peptides. Due to their physicochemical properties, these blockers are not suitable to study the physiological roles of SK channels in the central nervous system in vivo. Herein, we report the discovery of a chiral bis-tertiary amine with SK blocking properties from chemical modulation of laudanosine. AG525E1 has an affinity for SK channels (K(i)=293nM) approximately 100-fold higher than the tertiary compound laudanosine (K(i) approximately 30muM) and similar to the charged compound dequalinium (K(i)=221nM). AG525E1 equipotently blocks SK1, SK2 and SK3 currents in transfected cell lines. Because of its basic and lipophilic properties, it can reach central SK targets.

The sigma agonist 1,3-di-o-tolyl-guanidine directly blocks SK channels in dopaminergic neurons and in cell lines

Small conductance Ca(2+)-activated K(+) (SK) channels are widely expressed in the brain and underlie medium-duration afterhyperpolarizations (mAHPs) in many types of neurons. It was recently reported that the activation of sigma-1 (sigma(1)) receptors inhibits SK currents in rat hippocampus. Because many interactions between sigma receptors and brain dopaminergic systems have been reported, we set out to examine putative effects of sigma receptor ligands on the SK mediated mAHP in midbrain dopaminergic neurons. We found that 1,3-di-o-tolyl-guanidine (DTG) inhibited the mAHP in a concentration-dependent manner (approximately 60% inhibition at 100 microM), while other sigma receptor agonists (carbetapentane, (+)-SKF10047 and PRE-084) had little effect. Moreover, the effect of DTG was not affected by high concentrations of the sigma(1) receptor antagonist BD 1047. A role for sigma(2) receptors could also be excluded by the lack of effect of the sigma(2) receptor ligand 5-bromo-tetrahydroisoquinolinylbenzamide. These results argue against a coupling of sigma receptors to SK channels in dopaminergic neurons. We next hypothesized that DTG could directly block the channel. This hypothesis was tested in HEK-293 cells which were transiently transfected with rSK2 or hSK3 subunits. DTG inhibited the current flowing through both subtypes with mean IC(50)s approximately 200 microM. This action was also unaffected by BD 1047. Other sigma receptor ligands had little or no effect. We conclude that DTG directly blocks SK channels. This pharmacological action may be important to consider in future experimental settings.

Inhibition of KCa2.2 and KCa2.3 channel currents by protonation of outer pore histidine residues

Ion channels are often modulated by changes in extracellular pH, with most examples resulting from shifts in the ionization state of histidine residue(s) in the channel pore. The application of acidic extracellular solution inhibited expressed KCa2.2 (SK2) and KCa2.3 (SK3) channel currents, with KCa2.3 (pIC50 of ∼6.8) being approximately fourfold more sensitive than KCa2.2 (pIC50 of ∼6.2). Inhibition was found to be voltage dependent, resulting from a shift in the affinity for the rectifying intracellular divalent cation(s) at the inner mouth of the selectivity filter. The inhibition by extracellular protons resulted from a reduction in the single-channel conductance, without significant changes in open-state kinetics or open probability. KCa2.2 and KCa2.3 subunits both possess a histidine residue in their outer pore region between the transmembrane S5 segment and the pore helix, with KCa2.3 also exhibiting an additional histidine residue between the selectivity filter and S6. Mutagenesis revealed that the outer pore histidine common to both channels was critical for inhibition. The greater sensitivity of KCa2.3 currents to protons arose from the additional histidine residue in the pore, which was more proximal to the conduction pathway and in the electrostatic vicinity of the ion conduction pathway. The decrease of channel conductance by extracellular protons was mimicked by mutation of the outer pore histidine in KCa2.2 to an asparagine residue. These data suggest that local interactions involving the outer turret histidine residues are crucial to enable high conductance openings, with protonation inhibiting current by changing pore shape.

The interactions of apamin and tetraethylammonium are differentially affected by single mutations in the pore mouth of small conductance calcium-activated potassium (SK) channels

Valine residues in the pore region of SK2 (V366) and SK3 (V520) were replaced by either an alanine or a phenylalanine to evaluate the impact on the interactions with the allosteric blocker apamin. Unlike TEA which showed high sensitivity to phenylalanine mutated channels, the binding affinity of apamin to the phenylalanine mutants was strongly reduced. In addition, currents from phenylalanine mutants were largely resistant to block by apamin. On the other hand, when the valine residue was replaced by an alanine residue, an increase of the binding affinity and the amount of block by apamin was observed for alanine mutated SK2 channels, but not for mutated SK3 channels. Interestingly, the VA mutation reduced the sensitivity to TEA. In silico data confirmed these experimental results. Therefore, such mutations in the pore region of SK channels show that the three-dimensional structure of the SK tetramers can be disorganized in the outer pore region leading to reduced interaction of apamin with its target.