Steffensen Annette Buur, Bomholtz Sofia Hammami, Andersen Martin Nybo, Olsen Jesper Velgaard, Mutsaers Nancy, Lundegaard Pia Rengtved, Lundby Alicia, Schmitt Nicole
Danish National Research Foundation Centre for Cardiac Arrhythmia and Dept. of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Novo Nordisk Foundation Center for Protein Research, Department for Proteomics, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Cell Physiol Biochem. 2018;47(4):1742-1750. doi: 10.1159/000491007. Epub 2018 Jun 27.
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.
We employed mass spectrometry, whole-cell patch clamp electrophysiology, confocal microscopy, site-directed mutagenesis, and western blotting.
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.
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.
背景/目的：电压门控钾通道KV11.1最初是从大脑中克隆出来的，在多种组织中均有表达。磷酸化对通道功能的作用存在争议。在本研究中，我们旨在阐明蛋白激酶D介导的磷酸化程度及其作用。
我们采用了质谱分析、全细胞膜片钳电生理学、共聚焦显微镜、定点诱变和蛋白质印迹法。
利用大鼠和小鼠的脑组织，我们通过液相色谱-质谱联用技术（LC-MS）确定了KV11.1的几个磷酸化位点，并确定蛋白激酶D（PKD1）为可能的调节激酶。KV11.1与PKD1共表达可降低电流幅度，而不改变通道的蛋白水平或表面表达。基于体内实验和人胚肾293细胞（HEK293）实验的LC-MS结果，我们选择了四个KV11.1突变体进行进一步的功能分析。突变体S284A中假定的PKD磷酸化位点缺失增加了最大电流，表明S284是KV11.1中PKD的主要作用靶点。
我们的数据可能有助于缓解该领域关于蛋白激酶C（PKC）对KV11.1调节的长期争议。我们提出PKD1介导PKC对KV11.1的作用，并且我们发现PKD作用于通道N端的S284位点。
