Functions of the two types of calcium release channels in the sarcoplasmic reticulum of skeletal muscle.

在蛙骨骼肌的肌浆网(SR)中，两种类型的兰尼定受体(钙释放通道)的表达大致相同，分别对应于哺乳动物的1型和3型。本研究旨在阐明这两种通道各自的生理作用。我们取得了以下成果。(1)用只表达3型通道的1型基因敲除小鼠的骨骼肌检测3型通道的特性，发现有钙诱导的钙释放(CICR)活性，但需要更高的钙浓度才能激活。(2)建立了3基因敲除小鼠，其骨骼粘液表现为正常的EC偶联。(3)只有1型通道不能挽救1型基因敲除小鼠骨骼肌中受损的EC偶联，而2型和3型通道均不能。(4)我们已成功培育出1型和3型通道均被敲除的突变小鼠。这些小鼠骨骼肌的SR可以积累钙，但没有CICR，这表明在小鼠骨骼肌中表达的钙释放通道只有1型和3型，而不是2型。此外，还发现去极化诱导的钙释放是1型通道的特性。(5)钙调素除抑制CICR外，还在低钙激活浓度下促进CICR，而在高钙激活浓度下则表现出抑制作用。主要表现为对1型通道的增强作用和对3型通道的抑制作用。(6)Ryanodine不与关闭的通道结合，它通过生理刺激与开放的通道结合，并将其固定在开放状态以引起痉挛，此外，它还结合通过钙开放的通道(在CICR模式下开放)。因此，用放射性兰尼定测定生理收缩时1型和3型通道开放的相对大小是可能的。

In the sarcoplasmic reticulum (SR) of frog skeletal muscle, two types of ryanodine receptors (calcium release channels) , corresponding to mammalian type 1 and type 3, respectively, are expressed in about equal amount. This study is aiming at elucidation of the physiological role of each of these two types of channels. We have obtained the following results. (1) The properties of type 3 channels were examined by using skeletal muscles of type 1-knockout mice that expresses only type 3 channels, and found to show Ca-induced Ca release (CICR) activity but require a higher Ca concentration for activation. (2) We made type 3-knockout mice, the skeletal mucle of which showed normal EC coupling. (3) only type 1 channel but neither type 2 nor type 3 channels can rescue impaired EC coupling in skeletal muscle of type 1-knockout mice. (4)We have been able to make mutant mice in which both type 1 and type 3 channels are knocked out. The SR of skeletal muscle of these mice can accumulate Ca but CICR is absent, indicating that in mouse skeletal muscle the expressed Ca release channels are only types 1 and 3, but not type 2. It was also found that "depolarization" -induced Ca release is the property of type 1 channels. (5) Calmodulin was found to enhance CICR at low activating concentration of Ca in addition to inhibition of CICR so far reported that is shown at higher activating concentrations of Ca. It shows mainly an enhancing effect on type 1 channels and an inhibitory effect on type 3 channels. (6) Ryanodine, which does not bind to closed channels, binds to channels open by physiological stimulation and fixes in an open state to cause contracture, in addition to the wellknown fact that it binds channels open by Ca (open in CICR mode). Therefore, it will be possible by using radioactive ryanodine, to determine the relative magnitude of opening of type 1 and 3 channel in physiological contraction.