The KCNQ1 potassium channel is down-regulated by ubiquitylating enzymes of the Nedd4/Nedd4-like family.
OBJECTIVE
The voltage-gated KCNQ1 potassium channel regulates key physiological functions in a number of tissues. In the heart, KCNQ1 alpha-subunits assemble with KCNE1 beta-subunits forming a channel complex constituting the delayed rectifier current I(Ks). In epithelia, KCNQ1 channels participate in controlling body electrolyte homeostasis. Several regulatory mechanisms of the KCNQ1 channel complexes have been reported, including protein kinase A (PKA)-phosphorylation and beta-subunit interactions. However, the mechanisms controlling the membrane density of KCNQ1 channels have attracted less attention.


RESULTS
Here we demonstrate that KCNQ1 proteins expressed in HEK293 cells are down-regulated by Nedd4/Nedd4-like ubiquitin-protein ligases. KCNQ1 and KCNQ1/KCNE1 currents were reduced upon co-expression of Nedd4-2, the isoform among the nine members of the Nedd4/Nedd4-like family displaying the highest expression level in human heart. In vivo expression of a catalytically inactive form of Nedd4-2, able to antagonize endogenous Nedd4-2 in guinea-pig cardiomyocytes, increased I(Ks) significantly, but did not modify I(K1). Concomitant with the reduction in current induced by Nedd4-2, an increased ubiquitylation as well as a decreased total level of KCNQ1 proteins were observed in HEK293 cells. Pull-down and co-immunoprecipitation experiments showed that Nedd4-2 interacts with the C-terminal part of KCNQ1. The Nedd4/Nedd4-like-mediated regulation of the KCNQ1 channel complexes is strictly dependent on a PY motif located in the distal part of the C-terminal domain. When this motif was mutated, the current and ubiquitylation levels were unaffected by Nedd4-2, and Nedd4-2 proteins were neither pulled-down nor co-immunoprecipitated.


CONCLUSIONS
These results suggest that KCNQ1 internalization and stability is physiologically regulated by its Nedd4/Nedd4-like-dependent ubiquitylation. This mechanism may thereby be important in regulating the surface density of the KCNQ1 channels in cardiomyocytes and other cell types.
Jespersen
Thomas
T
Department of Pharmacology and Toxicology, University of Lausanne, Switzerland.
Membrez
Mathieu
M
Nicolas
Céline S
CS
Pitard
Bruno
B
Staub
Olivier
O
Olesen
Søren-Peter
SP
Baró
Isabelle
I
Abriel
Hugues
H
D000818:Animals; D002460:Cell Line; D015536:Down-Regulation; D056827:Endosomal Sorting Complexes Required for Transport; D015870:Gene Expression; D006168:Guinea Pigs; D006801:Humans; D051657:KCNQ1 Potassium Channel / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D000075702:Nedd4 Ubiquitin Protein Ligases; D018408:Patch-Clamp Techniques; D024642:Potassium Channels, Voltage-Gated / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
D056827:Endosomal Sorting Complexes Required for Transport; C489622:KCNE1 protein, human; D051657:KCNQ1 Potassium Channel; D024642:Potassium Channels, Voltage-Gated; D012333:RNA, Messenger; D025801:Ubiquitin; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases