Supplementary Material for: PKD Phosphorylation as Novel Pathway of KV11.1 Regulation

Background/Aims: The voltage-gated potassium channel KV11.1 has been originally cloned from the brain and is expressed in a variety of tissues. The role of phosphorylation for channel function is a matter of debate. In this study, we aimed to elucidate the extent and role of protein kinase D mediated phosphorylation. Methods: We employed mass spectrometry, whole-cell patch clamp electrophysiology, confocal microscopy, site-directed mutagenesis, and western blotting. Results: Using brain tissue from rat and mouse, we mapped several phosphorylated KV11.1 residues by LC-MS mass spectrometry and identified protein kinase D (PKD1) as possible regulatory kinase. Co-expression of KV11.1 with PKD1 reduced current amplitudes without altering protein levels or surface expression of the channel. Based on LC-MS results from in vivo and HEK293 cell experiments we chose four KV11.1 mutant candidates for further functional analysis. Ablation of the putative PKD phosphorylation site in the mutant S284A increased the maximal current indicating S284 as a main PKD target in KV11.1. Conclusions: Our data might help mitigating a long-standing controversy in the field regarding PKC regulation of KV11.1. We propose that PKD1 mediates the PKC effects on KV11.1 and we found that PKD targets S284 in the N-terminus of the channel.研究背景与目的：电压门控钾通道KV11.1最初从脑组织中克隆获得，可在多种组织中表达。磷酸化对该通道功能的调控作用长期存在争议。本研究旨在阐明蛋白激酶D（protein kinase D，PKD）介导的磷酸化过程的程度与调控作用。 实验方法：本研究采用质谱分析、全细胞膜片钳电生理学技术、共聚焦显微镜成像、定点突变技术以及蛋白质免疫印迹（western blotting）实验方法。 实验结果：通过大鼠和小鼠脑组织，利用液相色谱-质谱联用（LC-MS）技术定位了KV11.1上多个磷酸化氨基酸残基，并鉴定出蛋白激酶D1（PKD1）为其潜在的调控激酶。将KV11.1与PKD1共表达后，通道电流振幅显著降低，但未改变通道的蛋白表达水平与膜表面表达量。基于体内实验与人类胚胎肾293（HEK293）细胞实验的LC-MS结果，我们筛选出4个KV11.1突变体候选株用于后续功能分析。对预测的PKD磷酸化位点进行定点突变得到的S284A突变体，其最大电流振幅显著升高，表明S284是KV11.1上主要的PKD作用靶点。 结论：本研究数据有助于解决该领域长期以来关于蛋白激酶C（PKC）调控KV11.1的争议。我们提出PKD1介导了PKC对KV11.1的调控作用，且该激酶的作用靶点为通道N端的S284位点。