Abstract
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 contraction1. It has been shown by RT-PCR that BCM contains mRNA for four TRP channel homologues (TRPC 1, 3, 4 and 6), commonly regarded as molecular candidates for ROCs1. Here we tried to visually identify these TRPCs in the plasma membrane of BCM by immunofluorescence microscopy. A CCD fluorescence microimaging system (Olympus) was used. After 4-5 days culture of BCM cells on fibronectin-coated glass-bottomed dish in a serum-free HAM F12 media, the body of the cells was removed by gentle sonication under a hypotonic condition. The plasma membrane remaining attached on the glass surface was treated with polyclonal primary antibodies (2.5-5 μg/ml; Chemicon Co.) against putative cytoplasmic segments of the four TRPCs and visualized with secondary antibodies labelled with a fluorescent dye (Alexa Fluor 546; Invitrogen). The membrane preparations were also similarly immunostained with antibodies against MR3 and α-actin. All the anti-TRPC antibodies used gave many microscopically visible spots of immunofluorescence (roughly 1 spot/μm2) in the cell-free membrane preparation which was also positively stained with antibodies against MR3 and α-actin. The present results clearly demonstrate the existence of the four TRPC homologues in the plasma membrane of the BCM. 1. Takai et al (2004) J. Physiol. 559, 899-922 [Jpn J Physiol 55 Suppl:S134 (2005)]
