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.The muscarine-sensitive K ϩ current (M-current) 4 is a low threshold, non-inactivating K ϩ current that was first identified in sympathetic neurons (1) and is now known to be an important regulator of the excitability of many neuronal cell types in the central and peripheral nervous systems. The characteristic voltage and time dependence of the M-current leads to a "clamping" of the resting membrane potential upon activation of the current. The M-current therefore exerts a stabilizing effect on the resting membrane potential and attenuates neuronal excitability, thus limiting repetitive firing of neurons (2, 3). The M-current is mediated by members of the K v 7 family, which form a heterotetrameric channel consisting of KCNQ3 subunits associated with either KCNQ2 or KCNQ5 subunits, all containing six transmembrane domains (S1-S6), a short intracellular N-terminal segment, and a large intracellular C-terminal region (4 -8). Mutations in KCNQ2 and KCNQ3 are associated with epilepsy, in particular, benign neonatal familial seizures, and suppression of the M-current in conditional transgenic mice has been reported to cause spontaneous seizures (7, 9).At the single channel level, the M-current has been reported to be regulated by several receptor-mediated pathways and changes in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) (reviewed in Refs. 2 and 10); however, little is known about the factors that determine the cell surface density of the channels that underlie this current. One recently characterized mechanism for the regulation of cell surface levels of membrane proteins is ubiquitination by the Nedd4/ Nedd4-2 ubiquitin ligases, which results in the removal of the protein from the membrane (reviewed in Refs. ...

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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.

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