Function and regulation of TRPP2 ion channel revealed by a gain-of-function mutant.

Mutations in polycystin-1 and transient receptor potential polycystin 2 (TRPP2) account for almost all clinically identified cases of autosomal dominant polycystic kidney disease (ADPKD), one of the most common human genetic diseases. TRPP2 functions as a cation channel in its homomeric complex and in the TRPP2/polycystin-1 receptor/ion channel complex. The activation mechanism of TRPP2 is unknown, which significantly limits the study of its function and regulation. Here, we generated a constitutively active gain-of-function (GOF) mutant of TRPP2 by applying a mutagenesis scan on the S4-S5 linker and the S5 transmembrane domain, and studied functional properties of the GOF TRPP2 channel. We found that extracellular divalent ions, including Ca(2+), inhibit the permeation of monovalent ions by directly blocking the TRPP2 channel pore. We also found that D643, a negatively charged amino acid in the pore, is crucial for channel permeability. By introducing single-point ADPKD pathogenic mutations into the GOF TRPP2, we showed that different mutations could have completely different effects on channel activity. The in vivo function of the GOF TRPP2 was investigated in zebrafish embryos. The results indicate that, compared with wild type (WT), GOF TRPP2 more efficiently rescued morphological abnormalities, including curly tail and cyst formation in the pronephric kidney, caused by down-regulation of endogenous TRPP2 expression. Thus, we established a GOF TRPP2 channel that can serve as a powerful tool for studying the function and regulation of TRPP2. The GOF channel may also have potential application for developing new therapeutic strategies for ADPKD.

Extracellular Loops Are Essential for the Assembly and Function of Polycystin Receptor-Ion Channel Complexes.

Polycystin complexes, or TRPP-PKD complexes, made of transient receptor potential channel polycystin (TRPP) and polycystic kidney disease (PKD) proteins, play key roles in coupling extracellular stimuli with intracellular Ca2+ signals. For example, the TRPP2-PKD1 complex has a crucial function in renal physiology, with mutations in either protein causing autosomal dominant polycystic kidney disease. In contrast, the TRPP3-PKD1L3 complex responds to low pH and was proposed to be a sour taste receptor candidate. It has been shown previously that the protein partners interact via association of the C-terminal or transmembrane segments, with consequences for the assembly, surface expression, and function of the polycystin complexes. However, the roles of extracellular components, especially the loops that connect the transmembrane segments, in the assembly and function of the polycystin complex are largely unknown. Here, with an immunoprecipitation method, we found that extracellular loops between the first and second transmembrane segments of TRPP2 and TRPP3 associate with the extracellular loops between the sixth and seventh transmembrane segments of PKD1 and PKD1L3, respectively. Immunofluorescence and electrophysiology data further confirm that the associations between these loops are essential for the trafficking and function of the complexes. Interestingly, most of the extracellular loops are also found to be involved in homomeric assembly. Furthermore, autosomal dominant polycystic kidney disease-associated TRPP2 mutant T448K significantly weakened TRPP2 homomeric assembly but had no obvious effect on TRPP2-PKD1 heteromeric assembly. Our results demonstrate a crucial role of these functionally underexplored extracellular loops in the assembly and function of the polycystin complexes.

Detection of CXCR2 cytokine receptor surface expression using immunofluorescence.

The interleukin-8 (IL-8, CXCL8) chemokine, also known as the neutrophil chemotactic factor, is a cytokine that plays a key role in inflammatory response, cell proliferation, migration, and survival. IL-8 expression is increased not only in inflammatory disorders, but also in many types of cancer, including prostate cancer. IL-8 acts as a ligand for the C-X-C chemokine receptor 2 (CXCR2) protein present on the cell plasma membrane. Binding of the IL-8 ligand to the CXCR2 receptor results in an intracellular signaling pathway mediated by GTP binding proteins coupled to the receptor itself. Knowledge of the CXCR2 expression levels facilitates the understanding of the role and function of IL-8. In this chapter, we describe a protocol that uses the immunofluorescence method and confocal microscopy to analyze the CXCR2 surface expression in human prostate cancer cells. However, this protocol is easily adaptable to analyze the surface expression of other cytokine receptors in different cell types.