Supplementary Material for: PKD Phosphorylation as Novel Pathway of K_V11.1 Regulation

<b><i>Background/Aims:</i></b> The voltage-gated potassium channel K_V11.1 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. <b><i>Methods:</i></b> We employed mass spectrometry, whole-cell patch clamp electrophysiology, confocal microscopy, site-directed mutagenesis, and western blotting. <b><i>Results:</i></b> Using brain tissue from rat and mouse, we mapped several phosphorylated K_V11.1 residues by LC-MS mass spectrometry and identified protein kinase D (PKD1) as possible regulatory kinase. Co-expression of K_V11.1 with PKD1 reduced current amplitudes without altering protein levels or surface expression of the channel. Based on LC-MS results from <i>in vivo</i> and HEK293 cell experiments we chose four K_V11.1 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 K_V11.1. <b><i>Conclusions:</i></b> Our data might help mitigating a long-standing controversy in the field regarding PKC regulation of K_V11.1. We propose that PKD1 mediates the PKC effects on K_V11.1 and we found that PKD targets S284 in the N-terminus of the channel