Referenced in: none

Automatically associated channels: TRP , TRPC , TRPC6

Title: A disease-causing mutation illuminates the protein membrane topology of the kidney-expressed prohibitin homology (PHB) domain protein podocin.

Authors: Eva-Maria Schurek, Linus A Völker, Judit Tax, Tobias Lamkemeyer, Markus M Rinschen, Denise Ungrue, John E Kratz, Lalida Sirianant, Karl Kunzelmann, Martin Chalfie, Bernhard Schermer, Thomas Benzing, Martin Höhne

Journal, date & volume: J. Biol. Chem., 2014 Apr 18 , 289, 11262-71

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/24596097

Abstract
Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this membrane topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocin(P118L)) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2(P134S)). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. Podocin(P118L) and MEC-2(P134S) did not fractionate in detergent-resistant membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.