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.

Characterization of endogenous Kv1.3 channel isoforms in T cells

“…Besides V M regulation, Kv1.3‐mediated protein–protein interactions modulate the activity of various signaling pathways. Kv1.3 colocalizes with the TCR/CD3 complex in lipid rafts of Jurkat cells and redistributes to the IS formed with APC (Antigen Presenting Cells, Nicolaou et al, 2009; Panyi et al, 2004; Sebestyén et al, 2022). Activity of ion channels at the IS may alter the local ion composition in the restricted volume of the synaptic cleft, modulating the function of voltage‐gated channels and other molecules.…”

“…However, the functional role of these mechanisms (Kv1.3‐mediated membrane hyperpolarization vs. protein–protein interactions) or the role of V M ‐dependent changes in these interactions at the IS, is under debate. Although some studies link the immunomodulatory effects of Kv1.3 channels in T cells to their ability to modulate K + concentrations, because they were not reproduced with poreless mutant channels (Eil et al, 2016), other reports show that nonconducting Kv1.3 channels could recapitulate WT channel role in the IS formation (Sebestyén et al, 2022).…”

Characterization of endogenous Kv1.3 channel isoforms in T cells

“…Besides V M regulation, Kv1.3-mediated protein-protein interactions modulate the activity of various signaling pathways. Kv1.3 colocalizes with the TCR/CD3 complex in lipid rafts of Jurkat cells and redistributes to the IS formed with APC (Antigen Presenting Cells, Nicolaou et al, 2009;Panyi et al, 2004;Sebestyén et al, 2022). Activity of ion channels at the IS may alter the local ion composition in the restricted volume of the synaptic cleft, modulating the function of voltage-gated channels and other molecules.…”

“…protein-protein interactions) or the role of V M -dependent changes in these interactions at the IS, is under debate. Although some studies link the immunomodulatory effects of Kv1.3 channels in T cells to their ability to modulate K + concentrations, because they were not reproduced with poreless mutant channels (Eil et al, 2016), other reports show that nonconducting Kv1.3 channels could recapitulate WT channel role in the IS formation (Sebestyén et al, 2022).…”

Characterization of endogenous Kv1.3 channel isoforms in T cells.

“…When the membrane is depolarized, the S4 segment undergoes a conformational change, causing the S4-S5 linker to move and thus altering the opening or closing state of the conduction pathway (51). The intracellular NH2-and COOH-terminal domains participate in channel assembly and association with auxiliary subunits, as well as the regulation of inactivation and trafficking (52). Tetramerization of Kv1.3 subunits involves a conserved NH2-terminal domain (the T1 domain) (51,53).…”