Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking
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Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking. / Andersen, Martin Nybo; Hefting, Louise Leth; Steffensen, Annette Buur; Schmitt, Nicole; Olesen, Søren-Peter; Olsen, Jesper Velgaard; Lundby, Alicia; Rasmussen, Hanne Borger.
In: American Journal of Physiology: Cell Physiology, Vol. 309, No. 10, 15.11.2015, p. C693-C706.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Protein kinase A stimulates Kv7.1 surface expression by regulating Nedd4-2-dependent endocytic trafficking
AU - Andersen, Martin Nybo
AU - Hefting, Louise Leth
AU - Steffensen, Annette Buur
AU - Schmitt, Nicole
AU - Olesen, Søren-Peter
AU - Olsen, Jesper Velgaard
AU - Lundby, Alicia
AU - Rasmussen, Hanne Borger
N1 - Copyright © 2015, American Journal of Physiology - Cell Physiology.
PY - 2015/11/15
Y1 - 2015/11/15
N2 - 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 IKs current, which is enhanced by 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 co-assemble 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 lead 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.
AB - 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 IKs current, which is enhanced by 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 co-assemble 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 lead 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.
U2 - 10.1152/ajpcell.00383.2014
DO - 10.1152/ajpcell.00383.2014
M3 - Journal article
C2 - 26405101
VL - 309
SP - C693-C706
JO - American Journal of Physiology: Cell Physiology
JF - American Journal of Physiology: Cell Physiology
SN - 0363-6143
IS - 10
ER -
ID: 145720052