RT info:eu-repo/semantics/article
T1 Characterization of endogenous Kv1.3 channel isoforms in T cells
A1 Serna Pérez, Julia
A1 Peraza Pérez, Diego Alberto
A1 Moreno Estar, Sara
A1 Saez, Juan J.
A1 Gobelli, Dino Joaquin
A1 Simarro Grande, María
A1 Hivroz, Claire
A1 López López, José Ramón
A1 Cidad Velasco, María Del Pilar
A1 Fuente García, Miguel Ángel de la
A1 Pérez García, María Teresa
K1 Calcium signaling
K1 Electrophysiology
K1 Immunological synapse
K1 Kv1.3 channels
K1 T cells
K1 2407 Biología Celular
K1 24 Ciencias de la Vida
AB 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.
PB Wiley
SN 0021-9541
YR 2023
FD 2023
LK https://uvadoc.uva.es/handle/10324/58884
UL https://uvadoc.uva.es/handle/10324/58884
LA eng
NO Journal of Cellular Physiology, 2023.
NO Producción Científica
DS UVaDOC
RD 22-dic-2024