FIGURE 8.

USP36 regulates the protein levels and functions of Kv7.2/3 channels. A, USP36 competes with Kv7.2/3 binding to Nedd4-2. Recombinant protein GST-WW3 domain was incubated with a fixed amount of Kv7.2/3 channels and increasing amounts of USP36 and subjected to Western blotting analysis with different antibodies. A representative experiment is shown (n = 3). Note the displacement of Kv7.2/3 binding to GST-WW3 and the increased binding of USP36 when increasing amounts of USP36 were included. B, USP36 expression recues Kv7.2/3 down-regulation mediated by Nedd4-2. Lysates from HEK293 cells expressing Kv7.2/3 alone or in combination with GFP-Nedd4-2 and/or FLAG-USP36 were subjected to Western blotting analysis to detect the amount of Kv7.2/3 channels. GFP-Nedd4-2 and FLAG-USP36 protein levels from lysates are shown, and tubulin was used as a loading control. A representative experiment is shown (n = 3). Top and bottom asterisks indicate Kv7.2 and Kv7.3 subunits, respectively. Note that USP36 expression restores the levels of Kv7.2/3 channels in the presence of Nedd4-2. C, USP36 prevents Nedd4-2-mediated Kv7.2/3 current inhibition. Representative whole cell traces from HEK293T expressing Kv7.2/3, Nedd4-2, and USP36 proteins (top panels). D, normalized tail I-V relationships. The lines are fits of a Boltzmann equation to the data. E, graph showing the current densities of HEK293 cells expressing the corresponding constructs computed at −30 mV as the difference in quasi-instantaneous current after a prepulse to −110 mV (all channels closed) and +30 mV (all channels opened). The number of cells in each experiment is indicated in parentheses. The results are from two or more independent batches of cells and are shown as the means ± S.D. (unpaired Student's t test).