Apical Localization of a Functional TRPC3/TRPC6-Ca2+-Signaling Complex in Polarized Epithelial Cells

Extracellular Ca2+ Sensing in Salivary Ductal Cells

“…SMGs were excised from the animals and fixed in 10% formalin solution for 24 h for immunocytochemistry, dehydrated in graded concentrations of ethanol, embedded in paraffin, and used to prepare 5-10-m thick sections. Immunocytochemistry was performed on paraffin sections of mouse SMG as described previously (20). Briefly, sections were dewaxed, rehydrated, and permeabilized with 0.5% Triton X-100 non-ionic detergent, commonly used in phosphate-buffered saline (PBS), pH 7.5.…”

“…Anti-TRPC3 antibody was produced (rabbit polyclonal) and purified as described previously (20). All other specific chemicals and antibodies used are described in respective sections where they were used.…”

“…Fluorescence measurements were made using a Till Photonics-Polychrome IV spectrofluorometer and MetaFluor Imaging System (Universal Imaging Corporation). Fura-2 fluorescence in freshly dispersed submandibular cells was measured as described earlier (20,21) using an Olympus 50ϫ microscope, with an ORCA-ER camera (Hamamatsu) attached to a Polychrome IV (Till Photonics LLC). Ducts and acinar cells from the same glands were morphologically identified by their distinctive appearance under microscopic examination (22,23 Fully polarized monolayers were achieved at 72-96 h after cell plating.…”

“…RIPA buffer containing protease inhibitors was added directly onto the filter for cell lysis and frozen at Ϫ80°C for at least 2 h before use as described previously (20,26). Protein concentration was determined by using the Bio-Rad protein assay (microassay procedure).…”

Imbalance and dysfunction of transient receptor potential channels contribute to the pathogenesis of hypertension

“…These data implicate the concept of the formation of TRPC3/6 heteromeric complexes with a tightly receptor-operated TRPC6 subunit suppressing TRPC3 basal activity [41]. Furthermore, some multimerization data show that TRPC3/6 tetramers are formed in a heterologous expression system [42], in brain synaptosomes [43], and in polarized epithelial cells [44]. These findings may be compatible with the hypothesis that, in such heteromultimeric complexes, TRPC6 may suppress the basal activity of TRPC3, leading to a tightly receptor-regulated cation channel complex required for the physiological regu-lation of smooth muscle tone [40].…”