Role of C-Terminal Domain and Membrane Potential in the Mobility of Kv1.3 Channels in Immune Synapse Forming T Cells

Abstract

Voltage-gated Kv1.3 potassium channels are essential for maintaining negative membrane potential during T-cell activation. They interact with membrane-associated guanylate kinases (MAGUK-s) via their C-terminus and with TCR/CD3, leading to enrichment at the immunological synapse (IS). Molecular interactions and mobility may impact each other and the function of these proteins. We aimed to identify molecular determinants of Kv1.3 mobility, applying fluorescence correlation spectroscopy on human Jurkat T-cells expressing WT, C-terminally truncated (ΔC), and non-conducting mutants of mGFP-Kv1.3. ΔC cannot interact with MAGUK-s and is not enriched at the IS, whereas cells expressing the non-conducting mutant are depolarized. Here, we found that in standalone cells, mobility of ΔC increased relative to the WT, likely due to abrogation of interactions, whereas mobility of the non-conducting mutant decreased, similar to our previous observations on other membrane proteins in depolarized cells. At the IS formed with Raji B-cells, mobility of WT and non-conducting channels, unlike ΔC, was lower than outside the IS. The Kv1.3 variants possessing an intact C-terminus had lower mobility in standalone cells than in IS-engaged cells. This may be related to the observed segregation of F-actin into a ring-like structure at the periphery of the IS, leaving much of the cell almost void of F-actin. Upon depolarizing treatment, mobility of WT and ΔC channels decreased both in standalone and IS-engaged cells, contrary to non-conducting channels, which themselves caused depolarization. Our results support that Kv1.3 is enriched at the IS via its C-terminal region regardless of conductivity, and that depolarization decreases channel mobility.

Keywords: Kv1.3 channel; T cell; fluorescence correlation spectroscopy; immunological synapse; live cell; membrane depolarization; mobility.

Figure 1

Diffusion properties of WT and mutant Kv1.3 channels in standalone cells. (A) Diffusion coefficient of the slow component. The mobility of the wild type mGFP-tagged Kv1.3 channel (WT) was compared to that of non-conducting point mutant (NON-CON) and C-terminal deleted mutant (ΔC) Kv1.3 channels (empty boxes) expressed in Jurkat cells. The NON-CON mutant channel has the lowest mobility, and the ΔC mutant has the highest mobility. Depolarization by high external K⁺ concentration using high K⁺ solution decreased the mobility of the WT and ΔC channels significantly (filled boxes). (B) Average proportions (r_slow) of the slow component of the different Kv1.3 channels did not differ significantly. (C) Membrane potential of Jurkat cells expressing WT mGFP-Kv1.3 and the NON-CON mutant measured by patch clamp. On the box-and-whiskers plots, boxes represent the values of the 25th to 75th percentiles, whereas whiskers represent the 10th and 90th percentiles, the midline is the median value, and “+” marks the arithmetic mean. Mean values were compared with ANOVA and Student’s t-test. ** p < 0.01, **** p < 0.0001, n.s. not significant.

Figure 2

Mobility of MHC I heavy chain. Mobility of the MHC I glycoprotein was measured in standard solution in standalone Jurkat cells expressing WT, non-conductive point mutant (NON-CON), or C-terminal deleted mutant (ΔC) Kv1.3 channels. MHC I was labeled with Alexa546-W6/32 Fab. * p < 0.05, ** p < 0.01, n.s. not significant.

Figure 3

Transmission and fluorescence microscopic images of immunological synapses formed between a Raji B and Jurkat T cells expressing different mGFP-Kv1.3 variants. The red marks illustrate points inside and outside the IS where FCS measurements were typically carried out. Different versions of Kv1.3 are displayed: WT (A), NON-CON (B), and ΔC mutant (C). Panel (D) shows the extent of Kv1.3 enrichment at the IS according to Equation (3). The ratio of the average pixel intensity inside the IS and in the whole cell membrane was calculated from confocal images by using MATLAB (for details see Materials and Methods). * p < 0.05.

Figure 4

Mobility of wild type and mutant Kv1.3 channels on Jurkat T cells forming immunological synapses with Raji B cells at resting membrane potential and upon depolarization. (A) Slow diffusion coefficients of the WT and NON-CON channels decreased significantly inside the IS. However, that of the C-terminally truncated (ΔC) mutant did not change significantly. We also studied the effect of a depolarizing milieu. The mobility of WT and ΔC channels decreased significantly upon depolarization by high-K⁺ solution both inside and outside the IS. In contrast, the mobility of the NON-CON channels did not show significant alterations. (B) Average proportion of the slow diffusing component (r_slow) was not significantly affected by being partitioned in the IS, by mutation, or by depolarization. * p < 0.05, ** p < 0.01, **** p < 0.0001, n.s. not significant.

Figure 5

Mobility of IL-2 receptor α subunits labeled with Alexa 546-anti-Tac Fab on IS-forming Jurkat cells expressing WT mGFP-Kv1.3. IL-2Rα diffuses significantly more slowly in the IS than outside it, which may be the consequence of a more crowded environment. * p < 0.05, ** p < 0.01.

Figure 6

Distribution of Kv1.3 WT and F-actin in standalone and IS-engaged Jurkat cells. Kv1.3 was tagged with mGFP and F-actin was labeled with Alexa546-phalloidin. 3D reconstructions from z-stacks of confocal images are shown. (A) Standalone cells, (B) Jurkat cell in IS with Raji cell. The arrow marks the IS and the F-actin ring formed at its periphery.