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Its biophysical properties also resemble muscarinic cation current (m I in TRPC4-deficient mouse ileal myocytes).
Two most abundant TRPC4 variants are a “full-length” TRPC4α and shorter TRPC4β (or TRPC4Δ) lacking a stretch of 84 amino acids (781-864 in the case of mouse TRPC4) in the cytosolic C terminus (Δ84AA).
Integration of these stimuli might enable fine-tuning of channel activation as required for coordinated functions of the endothelium or smooth muscles in the intestine and the bladder, as well as neural signaling in the nervous system.
—HEK293 cells stably expressing mouse TRPC4 (m TRPC4) isoforms were grown under culture conditions as described (16) and seeded in 35-mm dishes 2 days prior to patch clamp recordings.
Here we show that TRPC4α but not TRPC4β was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP.
Its inhibitory action was dependent on the association of TRPC4α with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin.
For membrane potential measurements, the FLIPR membrane potential dye was diluted in the extracellular solution and added to cells following the manufacturer's protocol (Molecular Devices).
To control protein loading, the filter was stripped and incubated in the presence of a Ca Vβ2 antibody that recognizes the type 2 β subunit of voltage-activated Ca—Cell lysates from untransfected HEK293 cells and stably transfected cell lines were immunoprecipitated using goat anti-actin and goat anti-ezrin antibodies (both were from Santa Cruz Biotechnologies) in a buffer containing 150 m EDTA, and 0.5% Triton X-100, p H 7.4.PIP TRP and TRPL (d TRPs) both structurally and functionally as they are commonly gated by phospholipase C (PLC) activation (1).However, the molecular scenarios of TRPC activation downstream of PLC are diverse and may involve relevant lipids such as diacylglycerol (DAG) in the case of TRPC2, -C3, -C6, and -C7, inositol 1,4,5-trisphosphate formation, and/or Ca store depletion in the case of TRPC1 and -C3 (1).Total RNA was extracted from ileal myocytes using TRIzol reagent (Invitrogen).Reverse transcription was performed using Super Script II reverse transcriptase (Invitrogen) and random hexamers.