Augmenting the activity of calcium-activated potassium channel KCa3.1 revitalizes T-cell function in the immunosuppressive tumor microenvironment

The tumor microenvironment impedes antitumor immunity. Specifically, necrotic/dying tumor cells release substantial amounts of intracellular K+, increasing local extracellular K+ concentrations ([K+]e) up to 40-50 mM. Using tumors from hepatocellular carcinoma patients (n=6), we found that infiltrating CD4+ T-cells gradually polarized into immunosuppressive Th2 and regulatory T-cell subsets towards the tumor core. This suggested a crucial role of [K+]e-rich environment in the tumor core in directing the T-cell lineage commitment. In addition, we detected significantly reduced levels of the calcium-activated potassium channel Kca3.1 in activated T-cells from patients with hepatocellular carcinoma compared to that from healthy individuals. Exposing T-cells in vitro to high-[K+]e (50 mM [K+]e) significantly inhibited their function in terms of proliferation, cytokine production, extravasation, and killing of target cancerous cells. Comprehensive molecular, metabolomic and bioinformatic analyses, and subsequent validation experiments revealed that high-[K+]e impaired T-cell receptor signalling and downstream glycolysis pathways. By electrophysiological screening of a panel of quinoline-derived compounds using whole-cell patch-clamp recording, we identified SKA-346 as a KCa3.1 selective activator. Molecular docking predicted the KCa3.1 C-terminus intracellular loop linking the S4-S5 transmembrane segments as the SKA-346 binding site. Activating the KCa3.1 channel by SKA-346 (EC50 1.9 μM) significantly lowered T-cell intracellular K+ and revitalized their ability to effectively mount an antitumor response under high-[K+]e conditions. These findings suggest that SKA-346 could be combined with an immunotherapeutic drug to achieve better efficacy in treating cancer.