LAUSR

Similar to GABAA receptor-channels the calciummediated gating of the small-conductance KCa2 and the intermediate-conductance KCa3.1 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 calciumresponse curve of these voltage-independent, calmodulin-gated channels to the left and apparently increase their sensitivity to calcium, negative gating modulators decrease calcium sensitivity. However, in contrast to GABAA 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 KCa channels. One reason is of course the lower level of pharmacological interest. While GABAA receptors are firmly established as clinically used drug targets, no KCa2 or KCa3.1 channel modulators have yet reached the clinic despite their undeniable therapeutic potential for neurological, cardiovascular and inflammatory diseases. Another reason is the technical challenge involved in studying KCa channel gating. The gating apparatus is located at the intracellular C-terminus, where calmodulin, which functions as a calcium-sensing b-subunit, is constitutively associated with the calmodulin binding domain of the channels, 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, have been performed and it is currently hypothesized that KCa channel PAMs bind at the interface between the calmodulin N-lobe and the calmodulin-binding domain of the channels and thus “facilitate” mechanical opening ( D increased open channel probability) at a given Ca2C 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 KCa2.2, 3,4 or, more recently, by our own group using a combination of electrophysiology and site-directed mutagenesis. The latter study was guided by homology modeling of the KCa2.3 and KCa3.1 interface pocket and docking studies using the RosettaLigand computational modeling software. While the crystallography studies afforded the first insight into the atomistic mechanism of action of KCa activators, our molecular modeling study provides a plausible explanation for why KCa channel activators in general are 5–10-fold more potent in activating KCa3.1 than KCa2 channels. 5 The presence of R362 creates an extensive “background” hydrogen-bond network in the KCa3.1 interface pocket that stabilizes the main contacts NH2-substituted KCa activators make with M51 and E54 in calmodulin (Fig. 1). The three KCa2 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 KCa3.1