Orai1 Boosts SK3 Channel Activation

The interplay of SK3, a Ca2+ sensitive K+ ion channel, with Orai1, a Ca2+ ion channel, has been reported to increase cytosolic Ca2+ levels, thereby triggering proliferation of breast and colon cancer cells, although a molecular mechanism has remained elusive to date. We show in the current study, via heterologous protein expression, that Orai1 can enhance SK3 K+ currents, in addition to constitutively bound calmodulin (CaM). At low cytosolic Ca2+ levels that decrease SK3 K+ permeation, co-expressed Orai1 potentiates SK3 currents. This positive feedback mechanism of SK3 and Orai1 is enabled by their close co-localization. Remarkably, we discovered that loss of SK3 channel activity due to overexpressed CaM mutants could be restored by Orai1, likely via its interplay with the SK3–CaM binding site. Mapping for interaction sites within Orai1, we identified that the cytosolic strands and pore residues are critical for a functional communication with SK3. Moreover, STIM1 has a bimodal role in SK3–Orai1 regulation. Under physiological ionic conditions, STIM1 is able to impede SK3–Orai1 interplay by significantly decreasing their co-localization. Forced STIM1–Orai1 activity and associated Ca2+ influx promote SK3 K+ currents. The dynamic regulation of Orai1 to boost endogenous SK3 channels was also determined in the human prostate cancer cell line LNCaP.

Thio-ether functionalized glycolipid amphiphilic compounds reveal a potent activator of SK3 channel with vasorelaxation effect

Sulfur analogues of Ohmline feature activation properties of SK3 ion channels. One compound exhibits promising endothelium dependent vasorelaxation.

Hypoxia Promotes Prostate Cancer Aggressiveness by Upregulating EMT-Activator Zeb1 and SK3 Channel Expression

Hypoxia is a well-established feature of prostate cancer (PCa) and is associated with disease aggressiveness. The hypoxic microenvironment initiates multiple adaptive responses including epithelial-to-mesenchymal transition (EMT) and a remodeling of calcium homeostasis involved in cancer progression. In the present study, we identified a new hypoxia signaling pathway with a positive feedback loop between the EMT transcription factor Zeb1 and SK3, a Ca2+-activated K+ channel, which leads to amplifying store-operated Ca2+ entry. Zeb1 and SK3 channel were strongly upregulated by hypoxia both in vitro and ex vivo in organotypic cultures of human PCa. Taking into account the sensitivity of the SK3 channel to the membrane lipid composition, we identified lipids such as Ohmline (an alkyl ether lipid and SK3 inhibitor), linoleic acid (LA) and eicosapentaenoic acid (EPA) (fatty acids associated with indolent PCa), which were able to completely abrogate the hypoxia-induced changes in Zeb1 expression. Ultimately, better understanding of this new hypoxia-induced EMT pathway may allow to develop adjuvant therapeutic strategies, in order to control PCa aggressiveness and improve treatment outcomes.

Age-dependent neuroprotective effect of an SK3. channel agonist on excitotoxity to dopaminergic neurons in organotypic culture

We evaluated the age-dependency of the neuroprotective effect of an small-conductance calcium activated potassium channel 3 (SK3) agonist, 1-EBIO, on AMPA excitoxicity to dopaminergic neurons (DN) in organotypic cultures. Most TH+ neurons were also SK3+. SK3+/TH-cells (DN+) were common at each developmental stage but more prominently at day in vitro (DIV) 8. Young DN+ neurons were small bipolar and fusiform, whereas mature ones were large and multipolar. Exposure of organotypic cultures to AMPA (100 μm, 16 h) had no effect on the survival of DN+ at DIV 8, but caused significant toxicity at DIV 15 (n=15, p=0.005) and DIV 22 (n=15, p<<0.001). These results indicate that susceptibility of DN to AMPA excitotoxicity is developmental stage-dependent in embryonic VM organotypic cultures. Immature DN+ (small, bipolar) were increased after AMPA (100 μm, 16 h) at DIV 8, at the expense of the number of differentiated (large, multipolar) DN+ (p=0.039). This effect was larger at DIV 15 (p<<<0.0001) and at DIV 22 (p<<<0.0001). At DIV 8, 30 μM 1-EBIO resulted in a large increase in DN+. At DIV 15, AMPA toxicity was prevented by exposure to 30 μM, but not 100 μM 1-EBIO. At DIV 22, excitotoxicity was unaffected by 30 μM 1-EBIO, and partially reduced by 100 μM 1-EBIO. The effects of the SK3. channel agonist 1-EBIO on the survival of SK3.-expressing dopaminergic neurons were concentrationdependent and influenced by neuronal developmental stage.

Endothelial SK3 channel-associated Ca2+ microdomains modulate blood pressure

Activation of vascular endothelial small- (KCa2.3, SK3) or intermediate- (KCa3.1, IK1) conductance Ca2+-activated potassium channels induces vasorelaxation via an endothelium-derived hyperpolarization (EDH) pathway. Although the activation of SK3 and IK1 channels converges on EDH, their subcellular effects on signal transduction are different and not completely clear. In this study, a novel endothelium-specific SK3 knockout (SK3−/−) mouse model was utilized to specifically examine the contribution of SK3 channels to mesenteric artery vasorelaxation, endothelial Ca2+ dynamics, and blood pressure. The absence of SK3 expression was confirmed using real-time quantitative PCR and Western blot analysis. Functional studies showed impaired EDH-mediated vasorelaxation in SK3−/− small mesenteric arteries. Immunostaining results from SK3−/− vessels confirmed the absence of SK3 and further showed altered distribution of transient receptor potential channels, type 4 (TRPV4). Electrophysiological recordings showed a lack of SK3 channel activity, while TRPV4-IK1 channel coupling remained intact in SK3−/− endothelial cells. Moreover, Ca2+ imaging studies in SK3−/− endothelium showed increased Ca2+ transients with reduced amplitude and duration under basal conditions. Importantly, SK3−/− endothelium lacked a distinct type of Ca2+ dynamic that is sensitive to TRPV4 activation. Blood pressure measurements showed that the SK3−/− mice were hypertensive, and the blood pressure increase was further enhanced during the 12-h dark cycle when animals are most active. Taken together, our results reveal a previously unappreciated SK3 signaling microdomain that modulates endothelial Ca2+ dynamics, vascular tone, and blood pressure.