抄録
In bovine ciliary muscle (BCM) cells, stimulation of M3-muscarinic receptor (MR3) opens two types of receptor-operated non-selective cation channel (ROC) which serve as major pathways for Ca2+ entry during the tonic phase of contraction. It has been shown by RT-PCR that BCM contains mRNA for four TRP channel homologues (TRPC1, 3, 4, 6) commonly regarded as molecular candidates for ROCs. Here we tried to visually identify these TRPCs in the plasma membrane of BCM by immunofluorescence microscopy. After 3-5 days culture of BCM cells on fibronectin-coated glass in a serum-free media, the body of the cells was removed by gentle sonication under a hypotonic condition. The plasma membrane remaining attached on the glass was treated with primary antibodies against putative cytoplasmic segments of the four TRPCs and visualized with secondary antibodies labeled with a fluorescent dye. The membrane preparations were also similarly immunostained with antibodies against MR3, α-actin and Gαq/11. All the anti-TRPC antibodies used gave many microscopically visible spots of immunofluorescence (roughly 1 spots/μm2) in the cell-free membrane preparation which was also positively stained with antibodies against MR3 and α-actin. Anti-Gαq/11 antibodies used showed the dense distribution Gαq/11 proteins in or in the vicinity of the plasma membrane. The present results clearly demonstrate the existence of the four TRPC homologues, MR3, and Gαq/11 in the plasma membrane of the BCM. [J Physiol Sci. 2007;57 Suppl:S221]
