Molecular and Structural Mechanism of the Assembly of an Acid-Sensing Receptor/Ion Channel Complex

Abstract

Proteins in the polycystic kidney disease (PKD) family associate with subunits of the transient receptor potential polysystin (TRPP) subfamily to form functionally important complexes. For example, PKD1 and TRPP2 form a receptor/ion channel complex critical for kidney development and function, and mutations in both proteins cause human polycystic kidney disease. Another PKD member, PKD1L3, coassemble with TRPP3 to form a candidate sour taste receptor. The molecular mechanisms of the assembly of these complexes are largely unknown. Unlike TRP channel subunits, which contain six transmembrane segments, PKD proteins have 11 putative transmembrane segments, with a large extracellular N-terminus containing well-recognized protein-protein, protein-sugar, and protein-ligand interaction motifs. PKD proteins are therefore generally believed to function as cell surface receptors. In this study, we find that PKD1L3, when expressed in Xenopus oocytes, functions as a channel-forming subunit in an acid-sensing heteromeric channel complex formed by PKD1L3 and TRPP3. Single amino acid mutations in the putative pore region of both proteins alter the channel's permeability to monovalent and divalent cations. Single-molecule imaging analysis indicates that the PKD1L3/TRPP3 complex in the plasma membrane of live Xenopus oocytes contains one PKD1L3 and three TRPP3 subunits. We identify a C-terminal coiled-coil domain in TRPP3 and find it to be a trimer in solution and crystal structure. This coiled-coil domain trimer is critical for the assembly and surface expression of TRPP3 homomeric complexes and PKD1L3/TRPP3 heteromeric complexes. These results reinforce the notion that PKD and TRPP proteins associate with a 1:3 stoichiometry. They also provide evidence demonstrating that PKD proteins can function as channel-forming subunits.