ROMK1 channel activity is regulated by monoubiquitination

Abstract

The ubiquitination of proteins can signal their degradation, modify their activity or target them to specific membranes or cellular organelles. Here, we show that monoubiquitination regulates the plasma membrane abundance and function of the potassium channel, ROMK. Immunoprecipitation of proteins obtained from renal cortex and outer medulla with ROMK antibody revealed that this channel was monoubiquitinated. To determine the ubiquitin binding site on ROMK1, all intracellular lysine (Lys) residues of ROMK1 were individually mutated to arginine (Arg), and a twoelectrode voltage clamp was used to measure the ROMK1 channel activity in Xenopus oocytes. ROMK1 channel activity increased from 8.1 to 27.2 A only when Lys-22 was mutated to Arg. Furthermore, Western blotting failed to detect the ubiquitinated ROMK1 in oocytes injected with R1K22R. Patch-clamp experiments showed that biophysical properties of R1K22R were identical to those of wild-type ROMK1. Although total protein expression levels of GFP-ROMK1 and GFP-R1K22R in oocytes were similar, confocal microscopy showed that the surface fluorescence intensity in oocytes injected with GFP-R1K22R was higher than that of GFP-ROMK1. In addition, biotin labeling of ROMK1 and R1K22R proteins expressed in HEK293 cells showed increased surface expression of the Lys-22 mutant channel. Finally, expression of R1K22R in COS7 cells significantly stimulated the surface expression of ROMK1. We conclude that ROMK1 can be monoubiquitinated and that Lys-22 is an ubiquitin-binding site. Thus, monoubiquitination of ROMK1 regulates channel activity by reducing the surface expression of channel protein. This finding implicates the linking of a single ubiquitin molecule to channels as an important posttranslational regulatory signal.ubiquitination ͉ inwardly rectifying K channel (Kir.11) ͉ K recycling ͉ collecting duct ͉ thick ascending limb R OMK (KCNJ1) channel is an inwardly rectifying K channel located in the apical membrane of the thick ascending limb (TAL) and of the cortical collecting duct (CCD) (1). ROMK channels in the TAL are responsible for K recycling across the apical membrane and K recycling is essential for maintaining normal function of Na͞Cl͞K cotransporter (2). The importance of ROMK in K recycling in the TAL has been best demonstrated by the finding that loss-function mutations in ROMK cause salt wasting and metabolic alkalosis (Barter's syndrome) (3). ROMK is also responsible for K secretion in the CCD. Although the Ca 2ϩ -dependent maxi K channel has been shown to be involved in K secretion when urinary flow rate in the CCD is high (4, 5), ROMK channels play a principal role in K secretion under normal tubule flow (1, 2, 6).ROMK channel activity is regulated by hormones and dietary K intake by modulating the number of channels in the apical membrane (1,7,8). We and others have demonstrated that stimulation of V2 receptor with vasopressin or an increase in dietary K content increases the apical ROMK channel number in the CCD (9, 10). In contrast, low K intake decreas...

Chronic regulation of the renal Na+/H+exchanger NHE3 by dopamine: translational and posttranslational mechanisms

“…It was well documented that ENaC residence on the cell membrane and protein stability are regulated by ubiquitylation via the E3 ubiquitin ligase Nedd4-2 (96). In addition to ENaC, several other channels and transporters (ClC-5, CFTR, ROMK) have been found to be regulated by ubiquitylation (38,76,91). NHE1 ubiquitylation, endocytosis, and function are all regulated by Nedd4-1 and ␤-arrestin-1 (93).…”

Decoding ubiquitin sorting signals for clathrin-dependent endocytosis by CLASPs

“…This suggests that oligoubiquitylation is the rule rather than an exception in endocytosis. Although some membrane proteins do apparently undergo only monoubiquitylation (according to immunochemical detection), their multimerization ensures presentation of multiple ubiquitin molecules -for example, in the case of tetramerized ROMK1 potassium channels (Lin et al, 2005). Finally, monoubiquitin has a poor ability to promote endocytosis in Drosophila; this is evidenced by the markedly impaired downstream signalling of Delta in vivo following the replacement of its cytosolic domain with a single unextendible UbAllR (Wang and Struhl, 2004).…”

“…Notably, the ubiquitin-binding adaptors have surprisingly flexible recognition capability in higher eukaryotes in vivo, capturing both polymeric and oligomeric forms of ubiquitin that display different topologies (see above). This might explain the equally efficient internalization of oligomeric and monomeric plasma membrane proteins subject to monoubiquitylation, multiple-monoubiquitylation and/or polyubiquitylation, respectively; for example in ROMK1 (Lin et al, 2005), ENaC (Staub et al, 1997), EGF receptor (Haglund et al, 2003a;Huang et al, 2006;Mosesson et al, 2003), ␤ 2 adrenergic receptor-␤-arrestin complex (Shenoy et al, 2001), CD4 (Bartee et al, 2004), and MHC class I and B7.2 (Coscoy et al, 2001) (Fig. 1).…”

Regulation of ROMK (Kir1.1) channels: new mechanisms and aspects

“…However, the role of monoubiquitination in the regulation of membrane transporters and ion channels is not clear. Recently, it has been shown that ROMK channel activity could be regulated by monoubiquitination (31). First, immunoprecipitation of ROMK from renal tissue or from cell culture revealed a 50-kDa protein band that was also recognized by an ubiquitin antibody.…”