Departamento de Bioquímica y Biología Molecular y Fisiología, Universidad de Valladolid, Valladolid, Spain.
Unidad de Excelencia, Instituto de Biología y Genética Molecular (IBGM), CSIC, Valladolid, Spain.
J Cell Physiol. 2023 May;238(5):976-991. doi: 10.1002/jcp.30984. Epub 2023 Feb 28.
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 (V ), decreased Ca influx, and a reduction in the [Ca ] 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.
电压门控钾通道 Kv1.3 在 T 细胞激活中起关键作用；然而，缺乏可靠的抗体阻止了其在内源性条件下的准确检测。为了克服这一限制，我们创建了一个带有内源性 Kv1.3 通道标记的 Jurkat T 细胞系，以确定天然 Kv1.3 通道的表达、位置和激活后的变化。CRISPR-Cas9 技术用于在 KCNA3 基因的 C 末端插入 Flag-Myc 肽。使用 Western blot 分析和成像技术研究基础或激活的通道表达。我们鉴定了两种除经典通道（54 kDa）以外的 Kv1.3 同工型，它们在 N 末端不同：更长的同工型（70 kDa）和截断的同工型（43 kDa）。所有三种同工型在 T 细胞激活后均上调。我们专注于截断同工型（短形式，SF）的功能特征，因为它以前没有被描述过，并且可能存在于现有的 Kv1.3-/- 小鼠模型中。SF 在 HEK 细胞中的过表达引发了小幅度 Kv1.3 样电流，与经典 Kv1.3 不同，它不会诱导 HEK 增殖。为了在天然系统中探索内源性 SF 同工型的作用，我们生成了一个 Kv1.3 敲除 Jurkat 克隆和一个仅表达 SF 同工型的克隆。尽管经典同工型（长形式）主要位于质膜上，但 SF 仍保持在细胞内，在内核周围积累。因此，SF Jurkat 细胞没有显示 Kv1.3 电流，并且表现出去极化的静息膜电位（V ）、Ca 内流减少以及刺激时 [Ca ]增加减少。这些 Kv1.3 通道同工型的功能特征表明它们对参与免疫突触形成的信号通路有不同的贡献。我们得出结论，替代翻译起始在内源性 T 细胞中产生至少三种 Kv1.3 通道同工型，它们表现出不同的功能作用。对于其中一些功能，Kv1.3 蛋白不需要形成功能性质膜通道。
