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PCR was performed using sense primer 5′-CTGCAAATATCTCTGGGAAG-3′ and antisense primer 5′-CTACAATCTTGTGGTCACGTAGT-3′, for predicted products encompassing guinea pig TRPC4 c DNA derived from exons 5 to 9 and the coding sequence for Δ84AA with predicted products of 14 bp for the α and β isoforms, respectively.
—Microsomal membrane proteins were prepared from wild type mouse brain, TRPC4-deficient mouse brain, and smooth muscle cells isolated from guinea pig ileal longitudinal muscle.
The precipitated proteins were subjected to SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted using a polyclonal anti-TRPC4 antibody against an N-terminal peptide of TRPC4 (16).
—Control HEK293 cells and stable cell lines were seeded at 100,000 cells/well in wells of 96-well plates and allowed to grow for 18-24 h.
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).
—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.The functional implications of their differential tissue expression (13, 14) are intriguing, and they call for further investigations into the differential regulation of TRPC4 isoforms.They form heteromultimers whereby TRPC4α exerts a dominant negative effect ascribed to an autoinhibitory function of the Δ84AA region (15).The activation mechanism for the TRPC4/5 subgroup remains most elusive as none of the above appears to be involved (2).Notably, TRPC4/5 are most closely related to d TRPs, and recombinant d TRPL is known to be inhibited by the PLC substrate phosphatidylinositol 4,5-bisphosphate (PIP (5, 6), whereas the C termini of several TRPCs (C1, C5, C6, and C7) and TRPV1 bind to anionic lipids directly (7).
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.