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Serna, Julia; Peraza, Diego A.; Moreno‐Estar, Sara; Saez, Juan J.; Gobelli, Dino; Simarro, Maria; Hivroz, Claire; López‐López, José R.; Cidad, Pilar; Fuente, Miguel A.; Pérez‐García, M. Teresa
Journal of cellular physiology, 20/May , Letnik: 238, Številka: 5Journal Article
Voltage‐dependent potassium channel Kv1.3 plays a key role on T‐cell activation; however, lack of reliable antibodies has prevented its accurate detection under endogenous circumstances. To overcome this limitation, we created a Jurkat T‐cell line with endogenous Kv1.3 channel tagged, to determine the expression, location, and changes upon activation of the native Kv1.3 channels. CRISPR‐Cas9 technique was used to insert a Flag‐Myc peptide at the C terminus of the KCNA3 gene. Basal or activated channel expression was studied using western blot analysis and imaging techniques. We identified two isoforms of Kv1.3 other than the canonical channel (54 KDa) differing on their N terminus: a longer isoform (70 KDa) and a truncated isoform (43 KDa). All three isoforms were upregulated after T‐cell activation. We focused on the functional characterization of the truncated isoform (short form, SF), because it has not been previously described and could be present in the available Kv1.3−/− mice models. Overexpression of SF in HEK cells elicited small amplitude Kv1.3‐like currents, which, contrary to 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. Although the canonical isoform (long form) localizes mainly at the plasma membrane, SF remains intracellular, accumulating perinuclearly. Accordingly, SF Jurkat cells did not show Kv1.3 currents and exhibited depolarized resting membrane potential (VM), decreased Ca2+ influx, and a reduction in the Ca2+i increase upon stimulation. Functional characterization of these Kv1.3 channel isoforms showed their differential contribution to signaling pathways involved in formation of the immunological synapse. We conclude that alternative translation initiation generates at least three endogenous Kv1.3 channel isoforms in T cells that exhibit different functional roles. For some of these functions, Kv1.3 proteins do not need to form functional plasma membrane channels.
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JCR | SNIP | JCR | SNIP | JCR | SNIP | JCR | SNIP |
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