Inaugural Article: Podocin and MEC-2 bind cholesterol to regulate the activity of associated ion channels

The prohibitin homology (PHB)-domain proteins constitute a family of ≈1,800 proteins (SMART database; http://smart.embl-heidelberg.de) (1) all of which share an ≈150-aa domain similar to that in the mitochondrial protein prohibitin (2). More than 340 of these proteins, many of which have an N-terminal adjacent hydrophobic region that places them on the inner leaflet of the lipid bilayer, have been identified in animals. These membrane-associated proteins regulate osmotic homeostasis, mechanosensation, and cell signaling (3–5). Several of the animal PHB-domain proteins including flotillin, Podocin, prohibitin, stomatin, UNC-1, UNC-24, and the UNC-24-like mammalian protein SLP-1 are found in cholesterol-rich membrane fractions derived from the plasma membrane (reviewed in ref. 2). In this article, we investigate the function of these proteins using two members of the family, MEC-2 from Caenorhabditis elegans and Podocin from mouse. MEC-2 (6) and Podocin (7) have a single, central hydrophobic domain that embeds these proteins in the inner leaflet of the plasma membrane with their N- and C-terminal tails facing the cytoplasm (Fig. 1a). Although the two proteins contain different N and C termini, they have hydrophobic regions that are 35% identical and 75% similar and PHB-domains that are 50% identical and 80% similar (Fig. 1b). The PHB domain is critical for the action of both proteins (8, 9). The role of these conserved domains and the function of PHB-domain proteins, however, are unclear. Fig. 1. Structure and sequence of Podocin and MEC-2. (a) Membrane orientation of Podocin and MEC-2. The hydrophobic region (yellow) inserts into the inner leaflet of the plasma membrane, causing the remaining parts of the protein, including the PHB domain (red) . MEC-2 is part of a multiprotein-channel complex with the degenerin/epithelial Na+ channel (DEG/ENaC) proteins MEC-4 and MEC-10 that transduces gentle touch (6, 10, 11). In touch-receptor neurons, this channel complex is localized to regular puncta along the neuronal process; MEC-2 has been shown to regulate the MEC-4/MEC-10 ion channel in these puncta (9). However, the mechanism by which MEC-2 regulates ion-channel activity is elusive. Podocin localizes specifically to the slit diaphragm of the mammalian kidney. The slit-diaphragm is a specialized intercellular junction that connects adjacent foot processes of kidney podocytes and is part of the glomerular-filtration barrier (12). Slit diaphragm proteins induce signal transduction in podocytes, the visceral epithelial cells of the kidney glomerulus, that regulates cytoskeletal rearrangement and transcriptional activity (3). Like MEC-2, Podocin is part of a multiprotein complex containing the transmembrane proteins Neph1, Neph2, and Nephrin and the cytoplasmic adaptor protein CD2AP (3). Mutations in Podocin and the other proteins of this complex result in focal segmental glomerulosclerosis and congenital nephrotic syndrome, severe genetic kidney disorders in humans characterized by disruption of the filtration barrier (7, 13, 14). Recently, mutations in the transient receptor potential C channel protein TRPC6 were also shown to cause focal segmental glomerulosclerosis (15, 16). In this article, we show that Podocin and MEC-2 are cholesterol-binding proteins and that cholesterol binding plays an important role in regulating the activity of ion channels to which these PHB-domain proteins bind. Podocin, as we show here, binds to, colocalizes at the slit diaphragm with, and regulates the activity of TRPC6. Our results suggest that these proteins, similar to the proteins associated with MEC-2, may be part of a mechanosensitive protein complex at the slit diaphragm of podocytes. In general, we propose that many of the PHB-domain proteins regulate membrane protein function by binding sterols, perhaps by altering their local lipid environment.