Kv1.3 channel blockade with the Vm24 scorpion toxin attenuates the CD4+ effector memory T cell response to TCR stimulation

Background: In T cells, the K(v)1.3 and the K(Ca)3.1 potassium channels regulate the membrane potential and calcium homeostasis. Notably, during T-EM cell activation, the number of K(v)1.3 channels on the cell membrane dramatically increases. K(v)1.3 blockade results in inhibition of Ca2+ signaling in T-EM cells, thus eliciting an immunomodulatory effect. Among the naturally occurring peptides, the Vm24 toxin from the Mexican scorpion Vaejovis mexicanus is the most potent and selective K(v)1.3 channel blocker known, which makes it a promissory candidate for its use in the clinic. We have shown that addition of Vm24 to TCR-activated human T cells inhibits CD25 expression, cell proliferation and reduces delayed-type hypersensitivity reactions in a chronic inflammation model. Here, we used the Vm24 toxin as a tool to investigate the molecular events that follow K(v)1.3 blockade specifically on human CD4(+) T-EM cells as they are actively involved in inflammation and are key mediators of autoimmune diseases. Methods: We combined cell viability, activation, and multiplex cytokine assays with a proteomic analysis to identify the biological processes affected by K(v)1.3 blockade on healthy donors CD4(+) T-EM cells, following TCR activation in the presence or absence of the Vm24 toxin. Results: The peptide completely blocked K(v)1.3 channels currents without impairing T-EM cell viability, and in response to TCR stimulation, it inhibited the expression of the activation markers CD25 and CD40L (but not that of CD69), as well as the secretion of the pro-inflammatory cytokines IFN-gamma and TNF and the anti-inflammatory cytokines IL-4, IL-5, IL-9, IL 10, and IL-13. These results, in combination with data from the proteomic analysis, indicate that the biological processes most affected by the blockade of K(v)1.3 channels in a T cell activation context were cytokine-cytokine receptor interaction, mRNA processing via spliceosome, response to unfolded proteins and intracellular vesicle transport, targeting the cell protein synthesis machinery. Conclusions: The Vm24 toxin, a highly specific inhibitor of K(v)1.3 channels allowed us to define downstream functions of the K(v)1.3 channels in human CD4(+) TEM lymphocytes. Blocking K(v)1.3 channels profoundly affects the mRNA synthesis machinery, the unfolded protein response and the intracellular vesicle transport, impairing the synthesis and secretion of cytokines in response to TCR engagement, underscoring the role of K(v)1.3 channels in regulating TEM lymphocyte function.