SNF8, a member of the ESCRT-II complex, interacts with TRPC6 and enhances its channel activity.

BACKGROUND: Transient receptor potential canonical (TRPC) channels are non-selective cation channels involved in receptor-mediated calcium signaling in diverse cells and tissues. The canonical transient receptor potential 6 (TRPC6) has been implicated in several pathological processes, including focal segmental glomerulosclerosis (FSGS), cardiac hypertrophy, and pulmonary hypertension. The two large cytoplasmic segments of the cation channel play a critical role in the proper regulation of channel activity, and are involved in several protein-protein interactions. RESULTS: Here we report that SNF8, a component of the endosomal sorting complex for transport-II (ESCRT-II) complex, interacts with TRPC6. The interaction was initially observed in a yeast two-hybrid screen using the amino-terminal cytoplasmic domain of TRPC6 as bait, and confirmed by co-immunoprecipitation from eukaryotic cell extracts. The amino-terminal 107 amino acids are necessary and sufficient for the interaction. Overexpression of SNF8 enhances both wild-type and gain-of-function mutant TRPC6-mediated whole-cell currents in HEK293T cells. Furthermore, activation of NFAT-mediated transcription by gain-of-function mutants is enhanced by overexpression of SNF8, and partially inhibited by RNAi mediated knockdown of SNF8. Although the ESCRT-II complex functions in the endocytosis and lysosomal degradation of transmembrane proteins, SNF8 overexpression does not alter the amount of TRPC6 present on the cell surface. CONCLUSION: SNF8 is novel binding partner of TRPC6, binding to the amino-terminal cytoplasmic domain of the channel. Modulating SNF8 expression levels alters the TRPC6 channel current and can modulate activation of NFAT-mediated transcription downstream of gain-of-function mutant TRPC6. Taken together, these results identify SNF8 as a novel regulator of TRPC6.

Cross-Platform Comparison of Four Leading Technologies for Detecting EGFR Mutations in Circulating Tumor DNA from Non-Small Cell Lung Carcinoma Patient Plasma.

Analysis of circulating tumor DNA (ctDNA) is emerging as a powerful tool for guiding targeted therapy and monitoring tumor evolution in patients with non-small cell lung cancer (NSCLC), especially when representative tissue biopsies are not available. Here, we have compared the ability of four leading technology platforms to detect epidermal growth factor receptor (EGFR) mutations (L858R, exon 19 deletion, T790M and G719X) in ctDNA from NSCLC patients. Two amplification refractory mutation systems (cobas-ARMS and ADx-ARMS), a droplet digital polymerase chain reaction (ddPCR) and a next-generation sequencing (Firefly NGS) platform were included in the comparison. Fifteen EGFR mutations across twenty NSCLC patients were identified. Firefly NGS, cobas-ARMS and ddPCR all displayed superior sensitivity while ADx-ARMS was better suited for the qualitative detection of EGFR mutations with allele frequency higher than 1% in plasma and tissue samples. We observed high coincidence between the plasma and tissue EGFR mutational profiles for three driver mutations (L858R, exon 19 deletion and G719X) that are known targets of first generation EGFR-TKI therapies among patients who relapsed. Discrepancies between tissue and plasma EGFR mutational profiles were mainly attributable to spatial and temporal tumor heterogeneity, mutation inhibition due to therapy response and drug resistance (T790M). This study illustrates the challenges associated with selection of a technology platform for EGFR ctDNA analysis in the context of treatment evaluation and drug resistance detection.

Mechanism of alpha-latrotoxin action at nerve endings of neurohypophysis.

The neurotoxin alpha-latrotoxin elicits spontaneous exocytosis of neurotransmitter from neurons and peptide hormones from endocrine cells. While the mechanism of action is not fully understood, both Ca(2+)-dependent and Ca(2+)-independent pathways participate in the facilitation of release, with the relative contribution of the pathways differing among neuronal and endocrine cell types. Here, we investigate the actions of alpha-latrotoxin on neuroendocrine nerve endings that emanate from central nervous system neurons and, therefore, are unique in that they possess properties of central nerve endings and endocrine cells. Using intracellular [Ca(2+)] measurements both calcium-independent receptors for latrotoxin (CIRL or latrophilin) and neurexin 1 alpha receptors were found to be functionally present. Interaction of alpha-latrotoxin with these receptors stimulated secretion of vasopressin and oxytocin neuropeptide. The secretory response was entirely dependent upon toxin-mediated extracellular Ca(2+) influx, although alpha-latrotoxin also consistently triggered mobilization of Ca(2+) from an intracellular store. The mobilization of intracellular Ca(2+) relied on alpha-latrotoxin-mediated Na(+) influx and was blocked by the protonophore FCCP, thereby implicating mitochondria as the Ca(2+) store being mobilized. Using the whole cell recording configuration of the patch clamp, we report that alpha-latrotoxin interaction with the CIRL receptor on these nerve endings resulted in ionic pore formation, generating unitary inward current steps of 20 pA and a channel conductance of approximately 220 pS in Ca(2+)-free saline. Thus, alpha-latrotoxin stimulates Ca(2+)-dependent exocytosis in neurohypophysial nerve endings through receptor interaction and insertion of Ca(2+) permeable membrane pores. While alpha-latrotoxin mobilizes intracellular Ca(2+) stores the elevation in [Ca(2+)] reached is insufficient to trigger measurable exocytosis.

Inhibition of the TRPC5 ion channel protects the kidney filter.

An intact kidney filter is vital to retention of essential proteins in the blood and removal of waste from the body. Damage to the filtration barrier results in albumin loss in the urine, a hallmark of cardiovascular disease and kidney failure. Here we found that the ion channel TRPC5 mediates filtration barrier injury. Using Trpc5-KO mice, a small-molecule inhibitor of TRPC5, Ca2+ imaging in isolated kidney glomeruli, and live imagining of podocyte actin dynamics, we determined that loss of TRPC5 or its inhibition abrogates podocyte cytoskeletal remodeling. Inhibition or loss of TRPC5 prevented activation of the small GTP-binding protein Rac1 and stabilized synaptopodin. Importantly, genetic deletion or pharmacologic inhibition of TRPC5 protected mice from albuminuria. These data reveal that the Ca2+-permeable channel TRPC5 is an important determinant of albuminuria and identify TRPC5 inhibition as a therapeutic strategy for the prevention or treatment of proteinuric kidney disease.

Antagonistic regulation of actin dynamics and cell motility by TRPC5 and TRPC6 channels.

The Rho family of small guanosine triphosphatases (Rho GTPases: RhoA, Cdc42, and Rac1) regulates many aspects of cell behavior, including actin dynamics and cell migration. The generation of calcium ion (Ca(2+)) microdomains is critical in promoting cell migration because they control the localized activity of Rho GTPases. We identified receptor-activated TRPC5 and TRPC6 (transient receptor potential canonical type 5 and 6) channels as antagonistic regulators of actin remodeling and cell motility in fibroblasts and kidney podocytes. We show that TRPC5 is in a molecular complex with Rac1, whereas TRPC6 is in a molecular complex with RhoA. TRPC5-mediated Ca(2+) influx induces Rac1 activation, thereby promoting cell migration, whereas TRPC6-mediated Ca(2+) influx increases RhoA activity, thereby inhibiting cell migration. Our data unveil antagonistic Ca(2+) influx pathways as a conserved signaling mechanism for the integrated regulation of cell migration.

Taurine replacement attenuates hyperalgesia and abnormal calcium signaling in sensory neurons of STZ-D rats.

The etiology of painful diabetic neuropathy is poorly understood, but may result from neuronal hyperexcitability secondary to alterations of Ca2+ signaling in sensory neurons. The naturally occurring amino acid taurine functions as an osmolyte, antioxidant, Ca2+ modulator, inhibitory neurotransmitter, and analgesic such that its depletion in diabetes may predispose one to neuronal hyperexcitability and pain. This study reports the effects of taurine replacement on hyperalgesia and sensory neuron Ca2+ homeostasis in streptozotocin-diabetic (STZ-D) rats. Nondiabetic and STZ-D rats were treated with a 2% taurine-supplemented diet for 6-12 wk. Thermal hyperalgesia and mechanical allodynia were determined by measuring hindpaw withdrawal latency to radiant heat and the withdrawal threshold to the von Frey anesthesiometer. Intracellular Ca2+ signaling was explored in neurons from L4-L6 dorsal root ganglia (DRG), using fura 2 fluorescence. Taurine replacement of diabetic rats attenuated deficits of nerve conduction and prevented reductions of mechanical and thermal withdrawal threshold and latency, respectively. In small DRG sensory neurons from diabetic rats, recovery of intracellular Ca2+ concentration ([Ca2+]i) in response to KCl was slowed and 73% corrected by taurine. The amplitudes of caffeine and ATP-induced [Ca2+]i transients were decreased by 47 and 27% (P < 0.05), respectively, in diabetic rat DRG sensory neurons and corrected by 74 and 93% (P < 0.05), respectively, by taurine replacement. These data indicate that taurine is important in the regulation of neuronal Ca2+ signaling and that taurine deficiency may predispose one to nerve hyperexcitability and pain, complicating diabetes.