Polycystin-2 (PC2) is the product of the PKD2 gene, which is mutated in 10-15% patients of autosomal dominant polycystic kidney disease (ADPKD). PC2 is an integral transmembrane protein and acts as a calcium-permeable cation channel. The functional modulation of this channel by other protein partners remains largely unknown. In the present study, using a yeast two-hybrid approach, we discovered that both intracellular N- and C-termini of PC2 associate with alpha-actinins, actin-binding and actin-bundling proteins important in cytoskeleton organization, cell adhesion, proliferation and migration. The PC2-alpha-actinin association was confirmed by in vitro glutathione S-transferase pull-down and dot blot overlay assays. In addition, the in vivo interaction between endogenous PC2 and alpha-actinins was demonstrated by co-immunoprecipitation in human embryonic kidney 293 and Madin-Darby canine kidney (MDCK) cells, rat kidney and heart tissues and human syncytiotrophoblast (hST) apical membrane vesicles. Immunofluorescence experiments showed that PC2 and alpha-actinin were partially co-localized in epithelial MDCK and inner medullary collecting duct cells, NIH 3T3 fibroblasts and hST vesicles. We studied the functional modulation of PC2 by alpha-actinin in a lipid bilayer electrophysiology system using in vitro translated PC2 and found that alpha-actinin substantially stimulated the channel activity of reconstituted PC2. A similar stimulatory effect of alpha-actinin on PC2 was also observed when hST vesicles were reconstituted in lipid bilayer. Thus, physical and functional interactions between PC2 and alpha-actinin may play an important role in abnormal cell adhesion, proliferation and migration observed in ADPKD.

Polycystin-2 (PC2), encoded by the PKD2 gene, is mutated in 10-15% of autosomal dominant polycystic kidney disease (ADPKD) patients. PC2 is a Ca(2+)-permeable nonselective cation channel and is present in kidney and many other organs. Likewise, PKD2-mutated patients and mice exhibit extrarenal abnormalities. In comparison with cysts in the kidney, liver, and pancreas, abnormalities in the heart, brain, and vascular vessels are less understood. In particular, roles of PC2 in muscle and endothelia remain largely unknown. In the present study, using a yeast two-hybrid screening, we discovered that the PC2 carboxyl terminal domain (D682-V968) interacts with the cardiac troponin I, an important regulatory component of the actin microfilament in cardiac muscle cells. This interaction was demonstrated by GST pull-down and microtiter binding assays. Dose-dependent binding between PC2 and troponin I followed a Michaelis-Menten relationship, indicating a 1:1 binding stoichiometry. The interacting domains were located to the R872-H927 segment of PC2 and the M1-V107 and K106-L158 segments of troponin I. Co-immunoprecipitation experiments demonstrated that the cardiac and two skeletal isoforms of troponin I were all associated with PC2, when coexpressed in mouse fibroblast NIH 3T3 cells and Xenopus oocytes. Furthermore, reciprocal co-immunoprecipitation verified the interaction between the native polycystin-2 and troponin I in human adult heart tissues. This study thus provides new evidence for a direct attachment of PC2 to the actin microfilament network, in addition to the recently identified association between PC2 and trypomyosin-1. Troponin I functions as an inhibitory subunit of the troponin complex for calcium-dependent regulation of muscle contraction and as an inhibitor of angiogenesis seen in ADPKD. It is possible that altered interaction due to pathogenic polycystin-1 or -2 mutations can account for angiogenesis in ADPKD and may be corrected to some extent by exogenous troponin I.

Background: Pkd2L1 is a calcium-and acid-activated channel likely involved in acid sensing, but how it is regulated remains unclear. Results: Receptor for activated C kinase 1 (RACK1), known to regulate various receptors/channels, interacts with and inhibits the function of Pkd2L1. Conclusion: Pkd2L1 is a novel target channel of RACK1. Significance: Pkd2L1-RACK1 interaction may play important physiological roles through regulating channel activation by calcium or acid.

Transient receptor potential (TRP) polycystin 2 and 3 (TRPP2 and 3) are homologous members of the TRP superfamily of cation channels but have different physiological functions. TRPP2 is part of a flow sensor, and is defective in autosomal dominant polycystic kidney disease and implicated in left-right asymmetry development. TRPP3 is reported to implicate in sour tasting in bipolar cells of taste buds of the tongue and in the regulation of pH-sensitive action potential in neurons surrounding the central canal of spinal cord. TRPP3 is present in both excitable and non-excitable cells in various tissues, such as retina, brain, heart, testis, and kidney, but its common and cell type-specific functional characteristics remain largely unknown. In this study, we investigated physical and functional interactions between TRPP3 and a-actinin, an actinbundling protein known to regulate several types of ion channels. We employed planer lipid bilayer electrophysiology system to study the function of TRPP3 channel that was affinity-purified from Madin-Darby canine kidney cells. Upon reconstitution in bilayer, TRPP3 exhibited cation channel activities that were substantially augmented by a-actinin. The TRPP3-a-actinin association was documented by coimmunoprecipitation using native cells and tissues, yeast two-hybrid, and in vitro binding assays. Further, TRPP3 was abundantly present in mouse brain where it associates with aactinin-2. Taken together, a-actinin not only attaches TRPP3 to the cytoskeleton but also up-regulates TRPP3 channel function. It remains to be determined whether the TRPP3-aactinin interaction is relevant to acid sensing and other functions in neuronal and non-neuronal cells. Keywords: acid sensing, lipid bilayer electrophysiology, protein-protein interaction, tandem affinity purification, transient receptor potential polycystin 3, a-actinin.

The tandem affinity purification (TAP) procedure was initially developed as a tool for rapid purification of native protein complexes expressed at their natural levels in yeast cells. This purification procedure was also applied to study interactions between soluble proteins in mammalian cells. In order to apply this procedure to mammalian membrane proteins, we created a modified TAP tag expression vector and fused with the PKD2 gene, encoding a membrane cation channel protein, polycystin-2, mutated in 15% of autosomal dominant polycystic kidney disease. We generated epithelial Madin-Darby canine kidney cell line stably expressing TAP-tagged polycystin-2, improved the subsequent steps for membrane protein release and stability, and succeeded in purifying this protein. Using patch clamp electrophysiology, we detected specific polycystin-2 channel activities when the purified protein was reconstituted into a lipid bilayer system. Thus, this modified TAP procedure provides a powerful alternative to functionally characterize membrane proteins, such as ion channels, transporters and receptors, using cell-free system derived from mammalian cells.

Polycystin-L (PCL) is an isoform of polycystin-2, the product of the second gene associated with autosomal dominant polycystic kidney disease, and functions as a Ca(2+)-regulated nonselective cation channel. We recently demonstrated that polycystin-2 interacts with troponin I, an important regulatory component of the actin microfilament complex in striated muscle cells and an angiogenesis inhibitor. In this study, using the two-microelectrode voltage-clamp technique and Xenopus oocyte expression system, we showed that the calcium-induced PCL channel activation is substantially inhibited by the skeletal and cardiac troponin I (60% and 31% reduction, respectively). Reciprocal co-immunoprecipitation experiments demonstrated that PCL physically associates with the skeletal and cardiac troponin I isoforms in overexpressed Xenopus oocytes and mouse fibroblast NIH 3T3 cells. Furthermore, both native PCL and cardiac troponin I were present in human heart tissues where they indeed associate with each other. GST pull-down and microtiter binding assays showed that the C-terminus of PCL interacts with the troponin I proteins. The yeast two-hybrid assay further verified this interaction and defined the corresponding interacting domains of the PCL C-terminus and troponin I. Taken together, this study suggests that troponin I acts as a regulatory subunit of the PCL channel complex and provides the first direct evidence that PCL is associated with the actin cytoskeleton through troponin I.