The diverse effects of pathogenic point mutations on ion channel activity of a gain-of-function polycystin-2.

Autosomal dominant polycystic kidney disease is caused by mutations in PKD1 or PKD2 genes. The latter encodes polycystin-2 (PC2, also known as TRPP2), a member of the transient receptor potential ion channel family. Despite most pathogenic mutations in PKD2 being truncation variants, there are also many point mutations, which cause small changes in protein sequences but dramatic changes in the in vivo function of PC2. How these mutations affect PC2 ion channel function is largely unknown. In this study, we systematically tested the effects of 31 point mutations on the ion channel activity of a gain-of-function PC2 mutant, PC2_F604P, expressed in Xenopus oocytes. The results show that all mutations in the transmembrane domains and channel pore region, and most mutations in the extracellular tetragonal opening for polycystins domain, are critical for PC2_F604P channel function. In contrast, the other mutations in the tetragonal opening for polycystins domain and most mutations in the C-terminal tail cause mild or no effects on channel function as assessed in Xenopus oocytes. To understand the mechanism of these effects, we have discussed possible conformational consequences of these mutations based on the cryo-EM structures of PC2. The results help gain insight into the structure and function of the PC2 ion channel and the molecular mechanism of pathogenesis caused by these mutations.

The ion channel function of polycystin-1 in the polycystin-1/polycystin-2 complex.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2 gene, encoding the polycystic kidney disease protein polycystin-1 and the transient receptor potential channel polycystin-2 (also known as TRPP2), respectively. Polycystin-1 and polycystin-2 form a receptor-ion channel complex located in primary cilia. The function of this complex, especially the role of polycystin-1, is largely unknown due to the lack of a reliable functional assay. In this study, we dissect the role of polycystin-1 by directly recording currents mediated by a gain-of-function (GOF) polycystin-1/polycystin-2 channel. Our data show that this channel has distinct properties from that of the homomeric polycystin-2 channel. The polycystin-1 subunit directly contributes to the channel pore, and its eleven transmembrane domains are sufficient for its channel function. We also show that the cleavage of polycystin-1 at the N-terminal G protein-coupled receptor proteolysis site is not required for the activity of the GOF polycystin-1/polycystin-2 channel. These results demonstrate the ion channel function of polycystin-1 in the polycystin-1/polycystin-2 complex, enriching our understanding of this channel and its role in ADPKD.

A PKD1L3 splice variant in taste buds is not cleaved at the G protein-coupled receptor proteolytic site.

Mutations in polycystin proteins PKD1 and TRPP2 lead to autosomal dominant polycystic kidney disease. These two proteins form a receptor-ion channel complex on primary cilia. PKD1 undergoes an autoproteolysis at the N terminal G-protein-coupled receptor proteolytic site (GPS), which is essential for the function of PKD1. Whether GPS cleavage happens in other PKD proteins and its functional consequence has remained elusive. Here we studied the GPS cleavage of PKD1L3, a protein that associates with TRPP3 in taste cells and may play a role in sour taste. Our results show that PKD1L3 also undergoes GPS cleavage. Mutation at the GPS abolishes the cleavage, and the non-cleavable mutant does not traffic to the plasma membrane when associated with TRPP3. We also found that a splice variant of PKD1L3, which was originally identified in taste buds, is not cleaved. Amino acids L708 and S709, which are missing in this splice variant, are crucial for the GPS cleavage of PKD1L3 and the trafficking of the PKD1L3/TRPP3 complex. Our results gain insight into the molecular mechanism of the GPS cleavage of PKD1L3. The presence of the non-cleavable variant suggests the potential in vivo function of uncleaved PKD proteins.

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.

Knowledge, Attitude, Practices, and Preparedness of Dental Professionals in Prescribing Nicotine Replacement Therapy.

OBJECTIVE: To assess the knowledge, practice, attitude, and preparedness of dental professionals in prescribing nicotine replacement therapy (NRT). Methodology. A prevalidated voluntary web-based questionnaire was generated as a link through Google Drive and was sent to 117 dental professionals in North India using Whatsapp, Messenger, and Instagram social media platforms. A total of 94 responses were received and out of which 76 responses were analyzed (18 forms were excluded due to incomplete or duplicate responses). Frequency analysis was done using SPSS software version 21. RESULT: The participation rate was found to be 80.3%. More than half of the study population were familiar with the term NRT (77.6%) and its uses (67.1%), but approximately less than half of the total study subjects knew the duration (32.9%), cost (27.6%), dosage (25%), and contraindications (36.8%) of the NRT. Approximately 56.6% of the study participants showed a positive attitude towards helping patients to quit smoking through tobacco cessation counseling. Nearly one-fourth of the study population, i.e., 27.6%, were confident in explaining the negative impacts of tobacco, while 22.4% knew about the tobacco cessation protocol. Among the participants, only 27.6% reported that they practice NRT and out of which approximately less than 20% of the study participants were prescribing correct dose of NRT. CONCLUSION: Though study subjects had an ample knowledge regarding NRT use in tobacco cessation, it does not reflect their current attitude and preparedness. Thus, there is a need for continuing education to further train dental professionals for prescribing NRT.

Knowledge and Acceptance of the COVID-19 Vaccine for COVID-19 Disease Prevention among the Indian Population: A Mixed-Method Study.

Aim: To assess the Knowledge and Acceptance of the COVID vaccine among the Indian population. Materials and methods: The present mixed-method study was conducted in two phases. The first phase: quantitative assessment of knowledge and acceptance for the COVID-19 vaccine using an E survey (N = 606). The second phase: qualitative assessment using semi-structured face-to-face interviews with the study participants (N = 30) and assessment was done using a thematic approach. Study participants were selected using the convenience sampling method. Results: It was found that a large proportion of subjects in the 16−25 year of age group knew the cause of disease. But knowledge about its transmission process was found to be more in >60 years of age gap and almost all the participants in all the age group preferred Covishield. The vaccine acceptance rate was found to be low as compared to the knowledge. Conclusion: Most study participants were found to have satisfactory knowledge, but acceptance rate was comparatively lesser. Hence, more information and awareness campaigns must be launched reassuring the population about vaccine safety.

Analysis of the cell surface expression of cytokine receptors using the surface protein biotinylation method.

Cytokines are pleiotropic, low-molecular-weight proteins that regulate the immune responses to infection and inflammation. They stimulate the immune responses by binding to cytokine receptors on the cell plasma membrane. Thus, knowledge of the expression level of particular cytokine receptors on cell surface is crucial for understanding the cytokine function and regulation. One of the techniques to explore the membrane embedded cytokine receptors is cell surface biotinylation. Biotinylated surface proteins can be rapidly purified through the strong interaction between biotin and streptavidin. Here, we describe the procedure for surface biotinylation and purification of biotinylated cytokine receptors for further downstream analysis.

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.