Colocalization and nonrandom distribution of Kv1.3 potassium channels and CD3 molecules in the plasma membrane of human T lymphocytes

Abstract

Distribution and lateral organization of Kv1.3 potassium channels and CD3 molecules were studied by using electron microscopy, confocal laser scanning microscopy, and fluorescence resonance energy transfer. Immunogold labeling and electron microscopy showed that the distribution of FLAG epitope-tagged Kv1.3 channels (Kv1.3͞FLAG) significantly differs from the stochastic Poisson distribution in the plasma membrane of human T lymphoma cells. Confocal laser scanning microscopy images showed that Kv1.3͞FLAG channels and CD3 molecules accumulated in largely overlapping membrane areas. The numerical analysis of crosscorrelation of the spatial intensity distributions yielded a high correlation coefficient (C ‫؍‬ 0.64). A different hierarchical level of molecular proximity between Kv1.3͞FLAG and CD3 proteins was reported by a high fluorescence resonance energy transfer efficiency (E ‫؍‬ 51%). These findings implicate that reciprocal regulation of ion-channel activity, membrane potential, and the function of receptor complexes may contribute to the proper functioning of the immunological synapse.

Kv1.3 potassium channels are localized in the immunological synapse formed between cytotoxic and target cells

“…3 Top, red, Kv1.3͞FLAG labeled with anti-FLAG mAb followed by Alexa Fluor 546-RAMIG; green, MHC class I labeled with fluorescein-W6͞32 mAb; and blue, CD8 tagged by Alexa Fluor 647-MEM31 mAb). The distribution of Kv1.3 channels is similar to observations made on Jurkat cells by us (21) and by others (20). On the other hand, in CTLs conjugated with target cells, a rearrangement of Kv1.3͞FLAG was observed: in the majority of cases, ion channels were recruited into the CTL-target cell interface (Fig.…”

“…Our previous FRET studies on Jurkat cells indicated that Kv1.3͞FLAG molecules were in the molecular vicinity of CD3 (21). It was reported previously that CD3 molecules were also recruited into the IS formed between the CTL and the target cell (10).…”

“…Electron microscopic investigations revealed that the distribution of Kv1.3 channels in the plasma membrane of Jurkat T cells was nonrandom. Confocal laser-scanning microscopy (CLSM) images confirmed that Kv1.3 channels and CD3 molecules accumulated in largely overlapping membrane areas, whereas flow cytometric fluorescence resonance energy transfer (FRET) measurements proved a molecular proximity between Kv1.3 channels and CD3 proteins (21).…”

“…Bock et al (20) reported the redistribution of Kv1.3 channels between small sphingolipid-and cholesterol-enriched membrane rafts and large ceramide-enriched membrane platforms on increasing the ceramide content of the membrane. We have recently characterized the spatial distribution of Kv1.3 channels in the membrane of Jurkat T lymphocytes and the relationship between Kv1.3 channels and CD3 molecules (21). Electron microscopic investigations revealed that the distribution of Kv1.3 channels in the plasma membrane of Jurkat T cells was nonrandom.…”

“…The FLAG epitope was cloned into the gene encoding the human Kv1.3 potassium channel (Kv1.3͞FLAG; ref. 21) and CTLs were transfected with this gene by using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. Twenty-four hours after transfection, CTLs were washed in RPMI medium 1640 solution (without phenol red) and 2.5 ϫ 10 5 CTLs were mixed with equal amount of JY target cells in 1 ml of complete RPMI medium 1640 (without phenol red), were centrifuged (for 1 min at 200 ϫ g), and were kept at 37°C for 5 min.…”