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However, the nature of this inhibition remains unknown.In this study we found that PIP-dependent regulation involves cytoskeleton and TRPC4 C-terminal PDZ-interacting domain through which TRPC4/5 are known to associate with NHERF/ezrin-radixin-moesin (ERM)/actin cytoskeleton (16, 17).Protein samples (150 μg per lane) were separated on 8% SDS-polyacrylamide gels, blotted, and incubated in the presence of the TRPC4 antibody (14).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.Cells were washed with an extracellular solution and loaded with Fluo4-AM (2 μ was measured using a fluid handling integrated fluorescence plate reader, Flex Station (Molecular Devices).

Myenteric ganglia were removed from cell suspension using cell filtration through 80-μm nylon net filters (Millipore).Importantly, we found that several other phosphoinositides, which bind to the C termini in other TRPCs (7), including PI(3,4)P proteins, and perhaps other undefined components of PLC signaling.The multifaceted gating requirement suggests that TRPC4 channels are capable of integrating a variety of G-protein-dependent stimuli, including a PIP/cytoskeleton dependence much like the TRPC4-like muscarinic acetylcholine receptor (m ACh R)-activated cation channels in ileal myocytes.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).

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Its biophysical properties also resemble muscarinic cation current (m I in TRPC4-deficient mouse ileal myocytes).

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