The Ca²⁺-activated K⁺ channel KCa3.1 compartmentalizes in the immunological synapse of human T lymphocytes

Abstract

T cell receptor engagement results in the reorganization of intracellular and membrane proteins at the T cell-antigen presenting cell interface forming the immunological synapse (IS), an event required for Ca2+ influx. KCa3.1 channels modulate Ca2+ signaling in activated T cells by regulating the membrane potential. Nothing is known regarding KCa3.1 membrane distribution during T cell activation. Herein, we determined whether KCa3.1 translocates to the IS in human T cells using YFP-tagged KCa3.1 channels. These channels showed electrophysiological and pharmacological properties identical to wild-type channels. IS formation was induced by either anti-CD3/CD28 antibody-coated beads for fixed microscopy experiments or Epstein-Barr virus-infected B cells for fixed and live cell microscopy. In fixed microscopy experiments, T cells were also immunolabeled for F-actin or CD3ε, which served as IS formation markers. The distribution of KCa3.1 was determined with confocal and fluorescence microscopy. We found that, upon T cell activation, KCa3.1 channels localize with F-actin and CD3ε to the IS but remain evenly distributed on the cell membrane when no stimulus is provided. Detailed imaging experiments indicated that KCa3.1 channels are recruited in the IS shortly after antigen presentation and are maintained there for at least 15–30 min. Interestingly, pretreatment of activated T cells with the specific KCa3.1 blocker TRAM-34 blocked Ca2+ influx, but channel redistribution to the IS was not prevented. These results indicate that KCa3.1 channels are a part of the signaling complex that forms at the IS upon antigen presentation.

The Class II Phosphatidylinositol 3 kinase C2β Is Required for the Activation of the K+Channel KCa3.1 and CD4 T-Cells

“…Thus, one plausible hypothesis is that recruitment of NDPK-B, PI3K-C2␤, and KCa3.1 to peripheral microclusters in the IS is critical for the activation of KCa3.1. This is at least partly supported by a previous report showing that KCa3.1 is recruited to the IS, although the exact location of in the IS was not identified (Nicolaou et al, 2007). On the flip side, segregation of the negative regulators MTMR6 and PHPT1 away from peripheral microclusters would be one way of ensuring continuous signaling in the context of sustained TCR activation.…”

Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation

“…The two types of T lymphocyte potassium channel, Kv1.3 and KCa3.1, that are indirectly involved in functional Ca 2ϩ signaling by regulating the membrane potential (27) are also recruited to the IS (28 -30). Channel function and Ca 2ϩ signaling does not appear important for initial IS formation, because inhibiting either K ϩ channel with channel blockers (29,30) or CRAC channel function by expression of the nonconducting dominant-negative Orai1 subunit of the CRAC channel (SI Fig. 9) did not prevent molecular clustering in the contact zone.…”

The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to atherogenesis in mice and humans

“…The suppression of cytokine production might reduce the inflammatory processes within atherosclerotic lesions. The recent discovery that KCa3.1 traffics to the immune synapse during antigen presentation suggests that the channel plays an important role in early signaling events in T cells (42). Although the role of KCa3.1 channels in T cell migration has not been determined yet, this channel plays a role in the migration of other cells, including mast cells and macrophages (19,43,44).…”