Characterization of endogenous Kv1.3 channel isoforms in T cells

Resumen

The voltage-gated potassium channel Kv1.3 plays a crucial role in T-cell activation and is considered a promising target for the treatment of autoimmune diseases. However, the lack of reliable antibodies has prevented its accurate detection and study under endogenous conditions, so that most published studies have been conducted in heterologous systems. To address this limitation, we engineered a Jurkat T-cell line expressing endogenous Kv1.3 channels tagged with a signal peptide to investigate the expression and localization of native Kv1.3 channels, and their role associated to T cell physiological responses. Using the CRISPR-Cas9 tool, we inserted a Flag-Myc peptide at the C terminus of the KCNA3 gene. Basal and activated channel expression were assessed through western blot analysis and imaging techniques. Surprisingly, besides the canonical Kv1.3 channel (54 KDa), we identified two additional isoforms with distinct N termini: a longer isoform (70 KDa) and a truncated isoform (43 KDa). All three isoforms showed upregulation after T-cell activation. Our focus was on characterizing the truncated isoform (short form, SF) as it had not been previously described and could be present in available Kv1.3-/- mouse models. Overexpressing SF in HEK cells generated Kv1.3-like currents with smaller amplitudes, which, unlike canonical Kv1.3, did not induce HEK proliferation. To explore the role of endogenous SF isoform in a native system, we generated both a knockout Jurkat clone and a clone expressing only the SF isoform. While the canonical isoform localized primarily at the plasma membrane, SF remained intracellular, accumulating perinuclearly. Consequently, SF Jurkat cells lacked Kv1.3 currents, exhibited depolarized resting membrane potential (EM), reduced Ca2+ influx, and diminished increases in intracellular calcium ([Ca2+]i) upon stimulation. Functional characterization of these Kv1.3 channel isoforms revealed their differential contributions to signaling pathways involved in immunological synapse formation. In conclusion, alternative translation initiation generates at least three endogenous Kv1.3 channel isoforms in T cells with distinct functional roles. Importantly, some of these functions do not require the formation of functional plasma membrane channels by Kv1.3 proteins.