Submission ID 94025

Poster Code HR-P-45
Title of Abstract Structure-Activity study of the Transient Receptor Potential Canonical (TRPC6) Channels
Abstract Submission Introduction: Transient receptor potential canonical (TRPC6) channels play an important role in Ca2+ entry into several physiological and pathophysiological processes. Mutations in TRPC6 gene lead to focal segmental glomerulosclerosis, but their pathogenic mechanism remains elusive. TRPC6 gating modulation mechanism is still unknown. Recent results from our laboratory shown that Ca2+ and Mg2+ influence the structure of the C-terminal's helicoidal domain of TRPC6. These interactions are localized in the cytosolic part of the channel and may have an essential role in the regulation of TRPC6. Aims: To determine the binding site of Ca2+ and its role in the activation / regulation of TRPC6. Methods: By using the site-directed mutagenesis strategy, different mutants were generated, in the linker between the horizontal helical domain and the vertical helical domain, where aspartic acid residues were substituted by asparagine, glutamic acid residues were substituted by glutamine, and each of them were substituted by alanine, they were expressed in HEK293 cell. Then lipofection protocol was be optimized to make stably transfected with the Gαq-coupled M5 muscarinic receptor in HEK293 cells. Then, the [Ca2+]i was measured into the cells by Ca2+ imaging in real time using fura-2 Ca2+ probe. Then, the Ca2+ entry was evaluated using the Ca2+ depletion/re-addition protocol in fura-2 loaded cells after stimulation with carbachol (CCh), a muscarinic receptor agonist. Results: Ca2+ depletion was induced with 10 µM CCh in the Ca2+ free (0.5 mM EGTA) HBSS solution. Once the [Ca2+]i had returned to the basal level (3 min after the addition of CCh), the extracellular medium was repleted with 1.8 mm CaCl2. In the absence of extracellular Ca2+, CCh-induced Ca2+ release was similar whether cells were transfected with TRPC6WT or mutants TRPC6. After adding 1.8 mm CaCl2 to the external medium, [Ca2+]i rose by 28.9 nM under control conditions (no TRPC6 transfected) and
Please indicate who nominated you Sherbrooke university
What Canadian Institutes of Health Research (CIHR) institute is your research most closely aligned? Genetics
What Canadian Institutes of Health Research (CIHR) pillar of health research does your research fall under? Health systems services
PDF of abstract abstarct-JPhare 4.pdf
2023-03-03 at 12:56:06
Presenter and Author(s) Mahtab Keshvari
Mahtab Keshvari
Guylain Boulay

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