PKD Phosphorylation as Novel Pathway of KV11.1 Regulation

Abstract

Background/Aims: 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. 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 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 in vivo 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. Conclusions: 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.

Protein Kinase D1 Regulates Cardiac Hypertrophy, Potassium Channel Remodeling, and Arrhythmias in Heart Failure

“…PKD‐dependent regulation of K + channels has already been implicated in 2 previous studies 25 , 51 ; however, the exact contribution of PKD in modulating cardiac K + currents remained elusive. In diet‐induced obesity, upregulated PKD was associated with reduced CREB expression and downregulation of K V 1.5 and K V 2.1 channel expression in the heart.…”

“… 54 Indeed, PKD1 has been shown to regulate K V 11.1 (Kcnh2/human ether‐a‐go‐go‐related gene/ I Kr ) channels, which may involve the phosphorylation of S284 leading to reduced current amplitude, but interestingly without altering surface protein expression or the biophysical properties of the human ether‐a‐go‐go‐related gene channel expressed in human embryonic kidney‐293 cells. 51 Furthermore, the human ether‐a‐go‐go‐related gene promoter has been shown to be regulated by CREB. 25 Accordingly, we found PKD1‐dependent downregulation of I Kr and corresponding reduction in Kcnh2a and Kcnh2b expression post‐TAC (Figure S2 ; Figure 5C ).…”

A Novel Loss-of-Function Variant in the Chloride Ion Channel Gene Clcn2 Associates with Atrial Fibrillation

“…Western blotting. Cell lysates were prepared from three independent transfections as previously described 44 . Protein concentrations were determined by Bradford protein assays according to the manufacturer's instructions.…”

The Role and Mechanism of CRT0066101 as an Effective Drug for Treatment of Triple-Negative Breast Cancer

“…PRKDs have been reported in multiple cancer-promoting processes such as cell proliferation [8,9], cell migration [10,11], epithelial-mesenchymal transition [12], and angiogenesis [13]. Differences in function between PRKD isoforms have been described in breast, colon, gastric, and prostate cancers [10,[14][15][16][17][18][19][20]. In breast cancer, PRKD1 has been shown to promote cell growth [21] and inhibit invasiveness [22,23].…”