Molecular entity of regulatory mechanism of muscarinergic receptor operated cation channel

2014 Fiscal Year Final Research Report

• Project/Area Number: 24590266
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: General physiology
• Research Institution: Asahikawa Medical College
• Principal Investigator: TAKAI Akira 旭川医科大学, 医学部, 教授 (50126869)
• Co-Investigator(Kenkyū-buntansha): MIYAZU Motoi 旭川医科大学, 医学部, 講師 (40396346) ; TAKEYA Kosuke 旭川医科大学, 医学部, 助教 (20586862)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Keywords: ムスカリン受容体 / 細胞内信号伝達 / GTP結合蛋白 / 非選択性陽イオンチャネル / 毛様体筋 / 副交感神経 / 平滑筋 / 貯蔵量作動性カルシウムチャネル

Outline of Final Research Achievements

In bovine ciliary muscle (BCM), stimulation of M3-muscarinic receptors (M3R) opens two types of non-selective cation channel with different unitary conductances. In this work we developed a new method to obtain BCM cells with unprecedented quality and amount. The ciliary body dissected from bovine eye were treated with collagenase, and the dispersed cells were subjected centrifugation through discontinuous Percoll density-gradient of 1.050 and 1.060 g/mL. Cells were then collected from the 1.050/1.060 interface and cultured for 1-3 days. In the cultured BCM cells, carbachol (2 uM) evoked a phasic and tonic increase of [Ca2+]i. Caffeine (20 mM) caused a phasic rise of [Ca2+]i in the absence of extracellular Ca2+. These responses were clearly observed in 5 × 106 cells obtained by the single-step centrifugation procedure. Immunological staining revealed abundant expression of TRPC1, TRPC3, TRPC4, TRPC6 and Orai1 (as well as of M3R) in the plasma and endoplasmic membranes.

Free Research Field

生理学