The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid delayed rectifier K current (I) in human cells, plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. We previously reported that activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) induces endocytic degradation of cell-surface Kv1.5 channels, and a point mutation removing the phosphorylation site, T15A, in the N terminus of Kv1.5 abolished the PMA-effect. In the present study, using mutagenesis, patch clamp recording, Western blot analysis, and immunocytochemical staining, we demonstrate that ubiquitination is involved in the PMA-mediated degradation of mature Kv1.5 channels. Since the expression of the Kv1.4 channel is unaffected by PMA treatment, we swapped the N- and/or C-termini between Kv1.5 and Kv1.4. We found that the N-terminus alone did not but both N- and C-termini of Kv1.5 did confer PMA sensitivity to mature Kv1.4 channels, suggesting the involvement of Kv1.5 C-terminus in the channel ubiquitination. Removal of each of the potential ubiquitination residue Lysine at position 536, 565, and 591 by Arginine substitution (K536R, K565R, and K591R) had little effect, but removal of all three Lysine residues with Arginine substitution (3K-R) partially reduced PMA-mediated Kv1.5 degradation. Furthermore, removing the cysteine residue at position 604 by Serine substitution (C604S) drastically reduced PMA-induced channel degradation. Removal of the three Lysines and Cys604 with a quadruple mutation (3K-R/C604S) or a truncation mutation (Δ536) completely abolished the PKC activation-mediated degradation of Kv1.5 channels. These results provide mechanistic insight into PKC activation-mediated Kv1.5 degradation.

中文翻译：

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泛素化参与 PKC 介导的细胞表面 Kv1.5 通道降解


电压门控 Kv1.5 钾通道在人体细胞中传导超快速延迟整流 K 电流 (I)，在心房动作电位复极化和血管张力调节中发挥重要作用。我们之前报道过佛波醇 12-肉豆蔻酸酯 13-乙酸酯 (PMA) 激活蛋白激酶 C (PKC) 会诱导细胞表面 Kv1.5 通道的内吞降解，并且点突变会去除 N 末端的磷酸化位点 T15A Kv1.5 废除了 PMA 效应。在本研究中，利用诱变、膜片钳记录、蛋白质印迹分析和免疫细胞化学染色，我们证明泛素化参与了 PMA 介导的成熟 Kv1.5 通道的降解。由于 Kv1.4 通道的表达不受 PMA 处理的影响，因此我们交换了 Kv1.5 和 Kv1.4 之间的 N 端和/或 C 端。我们发现单独的 N 末端不会赋予 PMA 对成熟 Kv1.4 通道的敏感性，但 Kv1.5 的 N 末端和 C 末端确实赋予 PMA 对成熟 Kv1.4 通道的敏感性，表明 Kv1.5 C 末端参与通道泛素化。通过精氨酸取代（K536R、K565R 和 K591R）去除位置 536、565 和 591 处的每个潜在泛素化残基赖氨酸几乎没有影响，但通过精氨酸取代 (3K-R) 去除所有三个赖氨酸残基会部分减少 PMA -介导的Kv1.5降解。此外，通过丝氨酸取代（C604S）去除位置 604 的半胱氨酸残基大大减少了 PMA 诱导的通道降解。去除具有四重突变 (3K-R/C604S) 或截短突变 (Δ536) 的三个赖氨酸和 Cys604 完全消除了 PKC 激活介导的 Kv1.5 通道降解。这些结果提供了对 PKC 激活介导的 Kv1.5 降解的机制见解。