Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking

Abstract

The potassium channel Kv7.1 plays critical physiological roles in both heart and epithelial tissues. In heart, Kv7.1 and the accessory subunit KCNE1 forms the slowly activating delayed-rectifier potassium current current, which is enhanced by protein kinase A (PKA)-mediated phosphorylation. The observed current increase requires both phosphorylation of Kv7.1 and the presence of KCNE1. However, PKA also stimulates Kv7.1 currents in epithelial tissues, such as colon, where the channel does not coassemble with KCNE1. Here, we demonstrate that PKA activity significantly impacts the subcellular localization of Kv7.1 in Madin-Darby canine kidney cells. While PKA inhibition reduced the fraction of channels at the cell surface, PKA activation increased it. We show that PKA inhibition led to intracellular accumulation of Kv7.1 in late endosomes/lysosomes. By mass spectroscopy we identified eight phosphorylated residues on Kv7.1, however, none appeared to play a role in the observed response. Instead, we found that PKA acted by regulating endocytic trafficking involving the ubiquitin ligase Nedd4-2. We show that a Nedd4-2-resistant Kv7.1-mutant displayed significantly reduced intracellular accumulation upon PKA inhibition. Similar effects were observed upon siRNA knockdown of Nedd4-2. However, although Nedd4-2 is known to regulate Kv7.1 by ubiquitylation, biochemical analyses demonstrated that PKA did not influence the amount of Nedd4-2 bound to Kv7.1 or the ubiquitylation level of the channel. This suggests that PKA influences Nedd4-2-dependent Kv7.1 transport though a different molecular mechanism. In summary, we identify a novel mechanism whereby PKA can increase Kv7.1 current levels, namely by regulating Nedd4-2-dependent Kv7.1 transport.

Adrenergic Stress Protection of Human iPS Cell-Derived Cardiomyocytes by Fast Kv7.1 Recycling

“…Indeed, beat frequency-corrected FPDs were essentially invariant in stressed KCNQ1 -expressing cells compared to unstressed ones, which highlights the robustness of this adaptation system. Finally, our data are not at odds with previously published K v 7.1 trafficking mechanisms, notably RAB11-based slow recycling, as these may well play a role in ensuring homeostatic I Ks levels in cardiomyocytes or other contexts (Seebohm et al, 2007 ; Andersen et al, 2015 ). Likewise, our results are not redundant with those by Wang et al ( 2013 ) who proposed endoplasmic reticulum fragmentation as a slow-rate K v 7.1 trafficking mechanism.…”

“…RAB GTPases are key mediators of intracellular trafficking and have previously been associated with K v 7.1 recycling, albeit so far with mediating slow-rate ion channel turnover in non-cardiac cell contexts (Seebohm et al, 2007 ; Balse et al, 2012 ; Abbott, 2014 ; Andersen et al, 2015 ). hiPSC-CMs appeared to be refractory to the transfection of DNA constructs but we found that in-vitro transcribed mRNA could efficiently be delivered into these cells (Figure 5A ).…”

“…Another class of vesicles termed recycling endosomes are located in proximity to the plasma membrane allowing for fast fusion with selfsame to increase protein abundance at the cell surface. Small GTPases of the RAB protein family, the ubiquitin ligase NEDD4-2, as well as intracellular Ca 2+ elevation have been implicated in triggering K v 7.1/KCNE1 trafficking events in heterologous expression systems and animal models (Seebohm et al, 2007 ; Wang et al, 2013 ; Andersen et al, 2015 ). It is unclear, though, if these mechanisms represent the physiological reality in human cardiomyocytes, particularly under acute stress conditions.…”

The auxiliary subunit KCNE1 regulates KCNQ1 channel response to sustained calcium-dependent PKC activation

“…The C-terminus of KCNQ1 has been shown to interact with the C-terminus of KCNE1, and form a macromolecular complex with other molecules, including PI(4,5)P2, calmodulin, Nedd4.2 and AKAP-Yotiao, responsible for proper assembly, function and regulation of the channel [47][48][49][50][51]. KCNE1 expression has been shown to be important for regulation of the channel by PKA, PI(4,5)P2 and acute activation of the channel by PKC [6,7,13].…”

Regulation of Kv1.4 potassium channels by PKC and AMPK kinases

“…MDCK cells constitute an excellent cell system for studying trafficking of membrane proteins, such as ion channels [ 13-18 ]. The MDCK cells are, when grown to confluency, polarized cells with an apical and a basolateral membrane.…”