ABSTRACT
The electrical excitability of neurons is mediated primarily by voltage-gated ion channels, particularly voltage-gated Na(+) (Na(v)), K(+) (K(v)) and Cl(-) (ClC) channels. Cells regulate their electrical excitability by controlling not only the activity, but also the number of individual ion channels in the plasma membrane. There exist several mechanisms for regulating levels of voltage-gated ion channels: transcription and translation, retention and export from the endoplasmic reticulum as well as insertion and retrieval from the plasma membrane. Alterations in voltage-gated ion channel activity, composition and distribution can contribute to the pathophysiology of epilepsy, hypertension, neuropathic and inflammatory pain. One mechanism for retrieval is ubiquitination. Here specific ubiquitin ligases bind to membrane proteins to modulate and regulate their cellular fate. In this review, we focus on Nedd4 and Nedd4-2 ubiquitin ligases and the mechanisms by which they regulate voltage-gated ion channels and describe a novel paradigm on the mechanisms that underpin aberrant ion channel function in neurological disorders.
Subject(s)
Endosomal Sorting Complexes Required for Transport/metabolism , Ion Channel Gating , Ion Channels/metabolism , Neurons/enzymology , Protein Processing, Post-Translational , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Membrane/metabolism , Humans , Ion Channels/chemistry , Membrane Potentials , Models, Molecular , Nedd4 Ubiquitin Protein Ligases , Nervous System Diseases/metabolism , Protein Conformation , Protein Transport , Structure-Activity Relationship , UbiquitinationABSTRACT
The voltage-gated KCNQ2/3 and KCNQ3/5 K(+) channels regulate neuronal excitability. We recently showed that KCNQ2/3 and KCNQ3/5 channels are regulated by the ubiquitin ligase Nedd4-2. Serum- and glucocorticoid-regulated kinase-1 (SGK-1) plays an important role in regulation of epithelial ion transport. SGK-1 phosphorylation of Nedd4-2 decreases the ability of Nedd4-2 to ubiquitinate the epithelial Na(+) channel, which increases the abundance of channel protein in the cell membrane. In this study, we investigated the mechanism(s) of SGK-1 regulation of M-type KCNQ channels expressed in Xenopus oocytes. SGK-1 significantly upregulated the K(+) current amplitudes of KCNQ2/3 and KCNQ3/5 channels approximately 1.4- and approximately 1.7-fold, respectively, whereas the kinase-inactive SGK-1 mutant had no effect. The cell surface levels of KCNQ2-hemagglutinin/3 were also increased by SGK-1. Deletion of the KCNQ3 channel COOH terminus in the presence of SGK-1 did not affect the K(+) current amplitude of KCNQ2/3/5-mediated currents. Coexpression of Nedd4-2 and SGK-1 with KCNQ2/3 or KCNQ3/5 channels did not significantly alter K(+) current amplitudes. Only the Nedd4-2 mutant (S448A)Nedd4-2 exhibited a significant downregulation of the KCNQ2/3/5 K(+) current amplitudes. Taken together, these results demonstrate a potential mechanism for regulation of KCNQ2/3 and KCNQ3/5 channels by SGK-1 regulation of the activity of the ubiquitin ligase Nedd4-2.
Subject(s)
Immediate-Early Proteins/physiology , KCNQ Potassium Channels/physiology , KCNQ2 Potassium Channel/physiology , KCNQ3 Potassium Channel/physiology , Protein Serine-Threonine Kinases/physiology , Serum , Ubiquitin-Protein Ligases/physiology , Animals , Down-Regulation , Endosomal Sorting Complexes Required for Transport , Female , Ion Channel Gating , Nedd4 Ubiquitin Protein Ligases , Oocytes/drug effects , Oocytes/physiology , Rats , Recombinant Proteins/metabolism , Xenopus Proteins , Xenopus laevisABSTRACT
The muscarine-sensitive K(+) current (M-current) stabilizes the resting membrane potential in neurons, thus limiting neuronal excitability. The M-current is mediated by heteromeric channels consisting of KCNQ3 subunits in association with either KCNQ2 or KCNQ5 subunits. The role of KCNQ2/3/5 in the regulation of neuronal excitability is well established; however, little is known about the mechanisms that regulate the cell surface expression of these channels. Ubiquitination by the Nedd4/Nedd4-2 ubiquitin ligases is known to regulate a number of membrane ion channels and transporters. In this study, we investigated whether Nedd4/Nedd4-2 could regulate KCNQ2/3/5 channels. We found that the amplitude of the K(+) currents mediated by KCNQ2/3 and KCNQ3/5 were reduced by Nedd4-2 (but not Nedd4) in a Xenopus oocyte expression system. Deletion experiments showed that the C-terminal region of the KCNQ3 subunit is required for the Nedd4-2-mediated regulation of the heteromeric channels. Glutathione S-transferase fusion pulldowns and co-immunoprecipitations demonstrated a direct interaction between KCNQ2/3 and Nedd4-2. Furthermore, Nedd4-2 could ubiquitinate KCNQ2/3 in transfected cells. Taken together, these data suggest that Nedd4-2 is potentially an important regulator of M-current activity in the nervous system.
