Adenosine, a purine nucleoside, is present at high concentrations in tumors where it contributes to the failure of immune cells to eliminate cancer cells. The mechanisms responsible for the immunosuppressive properties of adenosine are not fully understood. We tested the hypothesis that adenosine’s immunosuppressive functions in human T lymphocytes are in part mediated via modulation of ion channels. The activity of T lymphocytes relies on ion channels. KCa3.1 and Kv1.3 channels control cytokine release and, together with TRPM7, regulate T cell motility. Adenosine selectively inhibited KCa3.1, but not Kv1.3 and TRPM7, in activated human T cells. This effect of adenosine was mainly mediated by A2A receptors as KCa3.1 inhibition was reversed by SCH58261 (selective A2A receptor antagonist), but not by MRS1754 (A2B receptor antagonist) and it was mimicked by the A2A receptor agonist CGS21680. Furthermore, it was mediated by the cAMP/PKAI signaling pathway as adenylyl-cyclase and PKAI inhibition prevented adenosine effect on KCa3.1. The functional implication of the effect of adenosine on KCa3.1 was determined by measuring T cell motility on ICAM-1 surfaces. Adenosine and CGS21680 inhibited T cell migration. Comparable effects were obtained by KCa3.1 blockade with TRAM-34. Furthermore, the effect of adenosine on cell migration was abolished by pre-exposure to TRAM-34. Additionally, adenosine suppresses IL-2 secretion via KCa3.1 inhibition. Our data indicate that adenosine inhibits KCa3.1 in human T cells via A2A receptor and PKAI thereby resulting in decreased T cell motility and cytokine release. This mechanism is likely to contribute to decreased immune surveillance in solid tumors.

Expression of CD1a protein defines a human dendritic cell (DC) subset with unique functional activities. We aimed to study the expression of the Nav1.7 sodium channel and the functional consequences of its activity in CD1a− and CD1a+ DC. Single-cell electrophysiology (patch-clamp) and quantitative PCR experiments performed on sorted CD1a− and CD1a+ immature DC (IDC) showed that the frequency of cells expressing Na+ current, current density, and the relative expression of the SCN9A gene encoding Nav1.7 were significantly higher in CD1a+ cells than in their CD1a− counterparts. The activity of Nav1.7 results in a depolarized resting membrane potential (−8.7 ± 1.5 mV) in CD1a+ IDC as compared with CD1a− cells lacking Nav1.7 (−47 ± 6.2 mV). Stimulation of DC by inflammatory signals or by increased intracellular Ca2+ levels resulted in reduced Nav1.7 expression. Silencing of the SCN9A gene shifted the membrane potential to a hyperpolarizing direction in CD1a+ IDC, resulting in decreased cell migration, whereas pharmacological inhibition of Nav1.7 by tetrodotoxin sensitized the cells for activation signals. Fine-tuning of IDC functions by a voltage-gated sodium channel emerges as a new regulatory mechanism modulating the migration and cytokine responses of these DC subsets.

Establishment of the immunological synapse (IS) between T lymphocytes and antigen-presenting cells is a key step in the adaptive immune response. Several proteins accumulate in the IS, such as the Kv1.3 potassium channel; however, the mechanism of this translocation is unknown. PSD-95 and SAP97 are adaptor proteins that regulate the polarized cell surface expression and localization of Kv1 channels in neurons. We investigated whether these proteins affect the redistribution of Kv1.3 into the IS in non-excitable human T cells. We show here that PSD-95 and SAP97 are expressed in Jurkat and interact with the C terminus of Kv1.3. Disruption of the interaction between PSD-95 or SAP97 and Kv1.3 in Jurkat was realized by the expression of a C-terminal truncated Kv1.3, which lacks the binding domain for these proteins, or by the knockdown of the expression of PSD-95 or SAP97 using specific shRNA. Expression of the truncated Kv1.3 or knockdown of PSD-95, but not the knockdown of SAP97, inhibited the recruitment of Kv1.3 into the IS; the fraction of cells showing polarized Kv1.3 expression upon engagement in an IS was significantly lower than in control cells expressing the full-length Kv1.3, and the rearrangement of Kv1.3 did not show time dependence. In contrast, Jurkat cells expressing the full-length channel showed marked time dependence in the recruitment into the IS peaking at 1 min after the conjugation of the cells. These results demonstrate that PSD-95 participates in the targeting of Kv1.3 into the IS, implying its important role in human T-cell activation.

Polarization of Kv1.3 channels is a necessary step in T-cell activation and motility. Nonetheless, the mechanisms regulating the Kv1.3 channel's membrane movement are not understood. This study provides evidence that cortactin, an actin-binding protein, controls Kv1.3 membrane motility via the channel's SH3 domain.