Are there superagonists for calcium-activated potassium channels?

KEYWORDS:

• EBIO
• gating modulator
• intermediate-conductance Ca²⁺-activated K⁺ channel
• K_Ca2.2
• K_Ca3.1
• NS309
• SKA-121
• SKA-31
• small-conductance Ca²⁺-activated K⁺ channel

This article refers to:

Similar to GABA_A receptor-channels the calcium-mediated gating of the small-conductance K_Ca2 and the intermediate-conductance K_Ca3.1 channels can be positively or negatively modulated by small molecule drugs, which, in analogy to the GABA field, have been termed positive (PAM) or negative allosteric modulators. While positive gating modulators like EBIO, NS309, SKA-31 and SKA-121 shift the calcium-response curve of these voltage-independent, calmodulin-gated channels to the left and apparently increase their sensitivity to calcium, negative gating modulators decrease calcium sensitivity.¹ However, in contrast to GABA_A receptors, where the binding site for the endogenous ligand GABA is located on the extracellular side and where allosteric modulation by benzodiazepines, neurosteroids and barbiturates has been studied in exquisite detail, only a small number of studies have been performed for K_Ca channels. One reason is of course the lower level of pharmacological interest. While GABA_A receptors are firmly established as clinically used drug targets, no K_Ca2 or K_Ca3.1 channel modulators have yet reached the clinic despite their undeniable therapeutic potential for neurological, cardiovascular and inflammatory diseases.¹ Another reason is the technical challenge involved in studying K_Ca channel gating. The gating apparatus is located at the intracellular C-terminus, where calmodulin, which functions as a calcium-sensing β-subunit, is constitutively associated with the calmodulin binding domain of the channels,² necessitating the performance of inside-out patch-clamp recordings when aiming to work at defined intracellular calcium concentrations. Nevertheless, a few studies, including some exquisite X-ray crystallography,^3,4 have been performed and it is currently hypothesized that K_Ca channel PAMs bind at the interface between the calmodulin N-lobe and the calmodulin-binding domain of the channels and thus “facilitate” mechanical opening ( = increased open channel probability) at a given Ca²⁺ concentration.

Both benzimidazole-type activators like EBIO and NS309 and naphthothiazole/oxazole-type activators like SKA-31 and SKA-121 (Fig. 1) have been shown to bind in this interface pocket either through co-crystallization of calmodulin in complex with the calmodulin-binding domain of K_Ca2.2,^3,4 or, more recently, by our own group using a combination of electrophysiology and site-directed mutagenesis.⁵ The latter study was guided by homology modeling of the K_Ca2.3 and K_Ca3.1 interface pocket and docking studies using the RosettaLigand computational modeling software. While the crystallography studies^3,4 afforded the first insight into the atomistic mechanism of action of K_Ca activators, our molecular modeling study provides a plausible explanation for why K_Ca channel activators in general are 5–10-fold more potent in activating K_Ca3.1 than K_Ca2 channels.⁵ The presence of R362 creates an extensive “background” hydrogen-bond network in the K_Ca3.1 interface pocket that stabilizes the main contacts NH₂-substituted K_Ca activators make with M51 and E54 in calmodulin (Fig. 1). The three K_Ca2 channels have shorter N or S residues in the corresponding position and therefore cannot form this hydrogen-bond network. The Rosetta models further suggested an explanation for why the 5-position methyl substituted SKA-121 is more potent on K_Ca3.1 and less potent on K_Ca2.3 than its parent compound SKA-31 by identifying an increased number of hydrophobic interactions in the “back” of the interface pocket for SKA-121 in the most frequently sampled lowest energy binding poses in K_Ca3.1.

Figure 1. Top, Chemical structures of the KCa channel activators and Rosetta model of SKA-121 (orange) docked into the interface between the K_Ca3.1 calmodulin-binding domain (blue) and calmodulin (yellow). See Brown et al.⁵ for details. Bottom, Cartoon of the effect of EBIO or NS309 on the calcium-response curve of K_Ca2.2 and of SKA-121 on the calcium-response curve of K_Ca3.1.

While these homology models are certainly helpful for explaining selectivity or for attempting structure based drug design, they fail to explain the experimentally observed ability of SKA-121 to further potentiate K_Ca currents at saturating Ca²⁺ concentrations. All previously published calcium-response curves for EBIO or NS309 on K_Ca2.2 show a “clean” left-ward shift without any increase in maximal effect (Fig. 1). SKA-121, in contrast, doubles K_Ca3.1 currents even in the presence of 10 μM of free intracellular calcium. This potentiation above the effect of the endogenous ligand, which is reminiscent of the superagonism observed on extrasynaptic GABA_A receptors, could potentially be explained by the assumed relatively low Ca²⁺-dependent P_o(max) of K_Ca3.1. However, it becomes harder to explain for K_Ca2.3, where SKA-121 is also still able to further potentiate currents in the presence of even 30 μM free calcium⁵ despite the fact that K_Ca2 channels are supposedly already fully open.

Future studies of K_Ca channel gating and the mechanism of action of K_Ca activators therefore will have to address several questions. First, how does the calmodulin mediated gating of the channels actually work? The dimer-of-dimers model suggested by the C-terminal crystal structures,^3,4,6 which all show two anti-parallel K_Ca2.2 fragments and two anti-parallel calmodulins forming a dimeric complex, has been questioned in favor of a model with four-fold rotational symmetry.⁷ This debate is unlikely to be resolved before a full-length structure of a K_Ca2 or K_Ca3.1 channel becomes available. Second, how do small molecules affect the gating and do they have the same effects on K_Ca3.1 and K_Ca2 channels? Up to now our laboratory is the only group that published K_Ca3.1 calcium-response curves in the presence of a K_Ca activator raising the question whether there are intrinsic differences between K_Ca3.1 and K_Ca2 channels, for which phosphatidylinositol 4,5-bisphosphate (PIP₂) has recently been shown to regulate channel activity by binding to the K_Ca2.2 calmodulin-binding domain/calmodulin complex.⁸ 3) Are there superagonists and partial agonists for K_Ca channels? And lastly, how different are K_Ca agonists that bind in the C-terminal interface pocket from K_Ca agonists¹ that bind in the pore domain?

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by the CounterACT Program, National Institutes of Health Office of the Director [U54NS079202], and the National Institute of Neurological Disorders and Stroke [R21NS101876].