The three small-conductance calcium-activated potassium (KCa2) channels and the related intermediate-conductance KCa3.1 channel are voltage-independent K+ channels that mediate calcium-induced membrane hyperpolarization. When intracellular calcium increases in the channel vicinity, it calcifies the flexible N lobe of the channel-bound calmodulin, which then swings over to the S4-S5 linker and opens the channel. KCa2 and KCa3.1 channels are highly druggable and offer multiple binding sites for venom peptides and small-molecule blockers as well as for positive- and negative-gating modulators. In this review, we briefly summarize the physiological role of KCa channels and then discuss the pharmacophores and the mechanism of action of the most commonly used peptidic and small-molecule KCa2 and KCa3.1 modulators. Finally, we describe the progress that has been made in advancing KCa3.1 blockers and KCa2.2 negative- and positive-gating modulators toward the clinic for neurological and cardiovascular diseases and discuss the remaining challenges.

Background: Dying tumor cells release intracellular potassium (K +), raising extracellular K + ([K + ] e) in the tumor microenvironment (TME) to 40-50 mM (high-[K + ] e). Here, we investigated the effect of high-[K + ] e on T cell functions. Materials and Methods: Functional impacts of high-[K + ] e on human T cells were determined by cellular, molecular, and imaging assays. Results: Exposure to high-[K + ] e suppressed the proliferation of central memory and effector memory T cells, while T memory stem cells were unaffected. High-[K + ] e inhibited T cell cytokine production and dampened antitumor cytotoxicity, by modulating the Akt signaling pathway. High-[K + ] e caused significant upregulation of the immune checkpoint protein PD-1 in activated T cells. Although the number of K Ca 3.1 calcium-activated potassium channels expressed in T cells remained unaffected under high-[K + ] e , a novel K Ca 3.1 activator, SKA-346, rescued T cells from high-[K + ] e-mediated suppression. Conclusion: High-[K + ] e represents a so far overlooked secondary checkpoint in cancer. K Ca 3.1 activators could overcome such ''ionic-checkpoint''-mediated immunosuppression in the TME, and be administered together with known PD-1 inhibitors and other cancer therapeutics to improve outcomes.

Intermediate-conductance (K Ca 3.1) and small-conductance (K Ca 2) calcium-activated K + channels are gated by calcium binding to calmodulin (CaM) molecules associated with the calmodulin binding domain (CaM-BD) of these channels. The existing K Ca activators like SKA-31, NS309and EBIO activate both channel types with similar potencies. In a previous chemistry effort we optimizes the benzothiazole pharmacophore of SKA-31 towards K Ca 3.1 selectivity and identified SKA-121 (5-methylnaphtho[2,1-d]oxazol-2-amine), which exhibits 40-fold selectivity for K Ca 3.1 over K Ca 2.3. In order to understand why introduction of a single CH 3 group in 5-position of the benzothiazole/oxazol system could achieve such a gain in selectivity for K Ca 3.1 over K Ca 2.3 we first localized the binding site of the benzothiazoles/oxazoles to the CaM-BD/CaM interface and then used the RosettaLigand computational modeling software to generate models of the K Ca 3.1 and K Ca 2.3 CaM-BD/CaM complexes with SKA-121. Based on a combination of mutagenesis and structural modeling we suggest that all benzothiazoles/oxazoles-type K Ca activators bind relatively "deep" in the CaM-BD/CaM interface and hydrogen-bond with E54 on CaM. In K Ca 3.1

Calcium-activated K+ channels constitute attractive targets for the treatment of neurological and cardiovascular diseases. To explain why certain 2-aminobenzothiazole/oxazole-type KCa activators (SKAs) are KCa3.1 selective we previously generated homology models of the C-terminal calmodulin-binding domain (CaM-BD) of KCa3.1 and KCa2.3 in complex with CaM using Rosetta modeling software. We here attempted to employ this atomistic level understanding of KCa activator binding to switch selectivity around and design KCa2.2 selective activators as potential anticonvulsants. In this structure-based drug design approach we used RosettaLigand docking and carefully compared the binding poses of various SKA compounds in the KCa2.2 and KCa3.1 CaM-BD/CaM interface pocket. Based on differences between residues in the KCa2.2 and KCa.3.1 models we virtually designed 168 new SKA compounds. The compounds that were predicted to be both potent and KCa2.2 selective were synthesized, and their activity and selectivity tested by manual or automated electrophysiology. However, we failed to identify any KCa2.2 selective compounds. Based on the full-length KCa3.1 structure it was recently demonstrated that the C-terminal crystal dimer was an artefact and suggested that the “real” binding pocket for the KCa activators is located at the S4-S5 linker. We here confirmed this structural hypothesis through mutagenesis and now offer a new, corrected binding site model for the SKA-type KCa channel activators. SKA-111 (5-methylnaphtho[1,2-d]thiazol-2-amine) is binding in the interface between the CaM N-lobe and the S4-S5 linker where it makes van der Waals contacts with S181 and L185 in the S45A helix of KCa3.1.

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Red cell volume is a major determinant of HbS concentration in sickle cell disease. Cellular deoxy-HbS concentration determines the delay time, the interval between HbS deoxygenation and deoxy-HbS polymerization. Major membrane transporter protein determinants of sickle red cell volume include the SLC12/KCC K-Cl cotransporters KCC3/SLC12A6 and KCC1/SLC12A4, and the KCNN4/KCa3.1 Ca2+-activated K+ channel (Gardos channel). Among standard inhibitors of KCC-mediated K-Cl cotransport, only [(dihydroindenyl)oxy]acetic acid (DIOA) has been reported to lack inhibitory activity against the related bumetanide-sensitive erythroid Na-K-2Cl cotransporter NKCC1/SLC12A2. DIOA has been often used to inhibit K-Cl cotransport when studying expression and regulation of other K+ transporters and K+ channels. We report here that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also abrogate activity of the KCNN4/KCa3.1 Gardos channel in human and mouse red cells and in human sickle red cells. DIOA inhibition of A23187-stimulated erythroid K+ uptake (Gardos channel activity) was chloride-independent and persisted in mouse red cells genetically devoid of the principal K-Cl cotransporters KCC3 and KCC1. DIOA also inhibited YODA1-stimulated, chloride-independent erythroid K+ uptake. In contrast, DIOA exhibited no inhibitory effect on K+ influx into A23187-treated red cells of Kcnn4-/- mice. DOIA inhibition of human KCa3.1 was validated (IC50 42 µM) by whole cell patch clamp in HEK-293 cells. RosettaLigand docking experiments identified a potential binding site for DIOA in the fenestration region of human KCa3.1. We conclude that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also block the KCNN4/KCa3.1 Gardos channel in normal and sickle red cells.

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In this study, we apply the canonical piecewise-linear (PWL) model to the modeling of super-resolution near-field structure (super-RENS) read-out signals because reliable and accurate channel modeling is essential for performance analysis and development of equalizers for super-RENS disc systems. To mitigate the nonlinear inter symbol interference (ISI), furthermore, we propose a canonical PWL model-based equalizer (PWLEQ) for super-RENS discs. The validity of the model and the equalizer is tested using radio frequency (RF) signal samples obtained from a super-RENS disc. The modeling experiment results verified that the canonical PWL model can be efficiently utilized for the nonlinear modeling of super-RENS systems. The raw bit error rate (BER) performance with the proposed equalizer is measured for various delays and step sizes of the canonical PWL model. We observe a marked improvement in raw BER when using the proposed equlaizer.