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

Abstract

BackgroundIn T cells, the Kv1.3 and the KCa3.1 potassium channels regulate the membrane potential and calcium homeostasis. Notably, during TEM cell activation, the number of Kv1.3 channels on the cell membrane dramatically increases. Kv1.3 blockade results in inhibition of Ca2+ signaling in TEM 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 Kv1.3 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 Kv1.3 blockade specifically on human CD4+ TEM cells as they are actively involved in inflammation and are key mediators of autoimmune diseases.MethodsWe combined cell viability, activation, and multiplex cytokine assays with a proteomic analysis to identify the biological processes affected by Kv1.3 blockade on healthy donors CD4+ TEM cells, following TCR activation in the presence or absence of the Vm24 toxin.ResultsThe peptide completely blocked Kv1.3 channels currents without impairing TEM 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-γ 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 Kv1.3 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.ConclusionsThe Vm24 toxin, a highly specific inhibitor of Kv1.3 channels allowed us to define downstream functions of the Kv1.3 channels in human CD4+ TEM lymphocytes. Blocking Kv1.3 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 Kv1.3 channels in regulating TEM lymphocyte function.Electronic supplementary materialThe online version of this article (10.1186/s12964-018-0257-7) contains supplementary material, which is available to authorized users.

Immunomagnetic separation is a suitable method for electrophysiology and ion channel pharmacology studies on T cells

“…This insensitivity of the blockers to the presence of the beads is different form the used of ~30 nm diameter gold particles conjugated to monoclonal antibodies targeting MHCI and MHC II molecules and the α subunit of the IL-2 receptor, where a slower association kinetics of a scorpion toxin (Pi2) and Kv1.3 was observed [ 22 ]. The insensitivity of Kv1.3 block to the presence of the beads might be important in physiological assays, where Kv1.3 blockers are widely used to inhibit the proliferation of various T cell subsets [ 52 , 53 ].…”

Kv1.3 Channel as a Key Therapeutic Target for Neuroinflammatory Diseases: State of the Art and Beyond

“…It is worth mentioning that Kv1.3 is the first K + channel to be identified outside electrically excitable tissues. Kv1.3 in T lymphocytes is responsible for controlling the membrane potential which is critical for the activation of these immune cells (Veytia-Bucheli et al, 2018). Several studies have confirmed that Kv1.3 is highly expressed in macrophages, microglia, and TEM cells, suggesting that Kv1.3 plays a crucial role in immune and inflammatory responses to human diseases such as multiple sclerosis (MS), rheumatoid arthritis, Type 1 diabetes, and asthma (Toldi et al, 2010;Huang et al, 2017;Tanner et al, 2017;Zhou et al, 2018).…”

Rescue of Retinal Degeneration in rd1 Mice by Intravitreally Injected Metformin

“…After considering the significance of the identified pathway differences and the number of related pathways, we found that the following five KEGG pathways were associated with the effects of metformin on rd1 mice: cytokine-cytokine receptor interactions, the TGF-β signaling pathway, the MAPK signaling pathway, the apoptosis pathway and the PI3K-Akt signaling pathway. It is widely accepted that cytokine-cytokine receptor interactions are associated with the inhibition of inflammation (Veytia-Bucheli et al, 2018), that the TGF-β signaling pathway is related to neuroprotection (Rodriguez-Martinez and Velasco, 2012) and that the activation of the MAPK signaling pathway and the PI3K-Akt signaling pathway can play a neuroprotective role (Zhu et al, 2016). Therefore, the results of KEGG pathway network mapping suggest that neuroprotective and anti-inflammatory effects may be important mechanisms underlying the ability of metformin to delay RP, consistent with our previous results.…”