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

Abstract

The prohibitin (PHB)-domain proteins are membrane proteins that regulate a variety of biological activities, including mechanosensation, osmotic homeostasis, and cell signaling, although the mechanism of this regulation is unknown. We have studied two members of this large protein family, MEC-2, which is needed for touch sensitivity in Caenorhabditis elegans, and Podocin, a protein involved in the function of the filtration barrier in the mammalian kidney, and find that both proteins bind cholesterol. This binding requires the PHB domain (including palmitoylation sites within it) and part of the N-terminally adjacent hydrophobic domain that attaches the proteins to the inner leaflet of the plasma membrane. By binding to MEC-2 and Podocin, cholesterol associates with ion-channel complexes to which these proteins bind: DEG͞ENaC channels for MEC-2 and TRPC channels for Podocin. Both the MEC-2-dependent activation of mechanosensation and the Podocin-dependent activation of TRPC channels require cholesterol. Thus, MEC-2, Podocin, and probably many other PHB-domain proteins by binding to themselves, cholesterol, and target proteins regulate the formation and function of large protein-cholesterol supercomplexes in the plasma membrane.prohibitin-domain proteins ͉ TRP channels ͉ DEG/ENaC channels ͉ slit diaphragm ͉ mechanosensation T he prohibitin homology (PHB)-domain proteins constitute a family of Ϸ1,800 proteins (SMART database; http:͞͞smart. embl-heidelberg.de) (1) all of which share an Ϸ150-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).

Podocyte Mitosis – A Catastrophe

“…Podocin may serve two closely related functions: the recruitment and/or stabilization of nephrin at the podocyte foot process, and the augmentation of nephrin signaling, perhaps by organizing specialized nephrin-containing microdomains [22]. In lipid microdomains, podocin clusters and regulates the transient receptor calcium channel (TRPC)-6 [23] and it has been suggested that this regulation gives the slit diaphragm the ability to act as a mechanosensor that enables the podocyte to remodel its cytoskeleton and contract its foot process in response to mechanical stimuli [24]. Finally, TRPC-6 is a critical controller of actin cytoskeleton.…”

Touch sensitivity in Caenorhabditis elegans

“…Stomatin, podocin, MEC-2, UNC-24, and several other PHB-domain proteins have a hydrophobic [44] region N-terminally adjacent to the PHB domain [3,8,39,77]. This domain in MEC-2, stomatin, and podocin attaches the proteins to the inner leaflet of the plasma membrane [40].…”

“…In MEC-2, the domain is required for homooligomerization, localization to puncta, and amplification of MEC-4d currents in frog oocytes [98]. Recently, MEC-2 has also been shown to bind cholesterol; this binding requires the PHB domain and five amino acids in the adjacent hydrophobic region [40]. In HEK 293T cells, expression of MEC-2 can bind and recruit cholesterol to rat αENaC (a DEG/ENaC protein whose TM2 domain can substitute for that of MEC-4 in vivo [36]), and MEC-2 probably plays a similar role in C. elegans touch receptor neurons.…”

“…In HEK 293T cells, expression of MEC-2 can bind and recruit cholesterol to rat αENaC (a DEG/ENaC protein whose TM2 domain can substitute for that of MEC-4 in vivo [36]), and MEC-2 probably plays a similar role in C. elegans touch receptor neurons. While cholesterol is not required for MEC-2 multimerization or association with DEG/ENaC proteins, gentle touch sensitivity in worms is dependent on sterols [40]. The PHB domain of UNC-24 has not been tested for the ability to bind cholesterol, but it is necessary for the association of UNC-24 with other channel subunits [98].…”

Expression of trpC1 and trpC6 orthologs in zebrafish

“…In mice and humans, TRPC6 protein is expressed in kidney podocytes in close proximity to the filtration slits of the glomerular filter. Recent studies support the notion that TRPC6-mediated calcium signaling contributes to the maintenance of the glomerular slit diaphragm and the regulation of glomerular permselectivity (Huber et al, 2006). TRPC6 knockout mice display defective vasomotor control and a sensitized myogenic response, suggesting an important role in smooth muscle contractility (Dietrich et al 2005, Weissmann et al, 2006.…”