Molecular Basis of Multiple Open States in Smooth Muscle Calcium Channels and Related Intracellular Signalling

已知一些平滑肌同时具有低电压激活(LVA)和高电压激活(HVA)Ca 2 通道电流，并且HVA电流分量在所有平滑肌中占主导地位。之前，我们提出，大去极化过程中Ca^2通道构象从正常开放状态（O1）到第二开放状态（O2）的转变与Ca^2通道激动剂诱导的长通道开放不同，并且这两种机制是分开运作的。结果，DHP Ca^<2>通道激动剂和去极化的组合在天然平滑肌Ca^<2>通道中产生至少四种开放状态。在本研究项目“平滑肌钙通道和相关细胞内信号传导中多个开放状态的分子基础”的任期内，我们首先检查了多个开放状态模型是否可以系统地解释“U形失活”和“缓慢”的特征。使用膜片钳技术，CHO 细胞中的“失活”仅抑制 L 型 Ca^<2> 通道 (Ca,1.2b) 的平滑肌 _1 亚基。实验表明，单独的平滑肌_1亚基可以再现“U形失活”和“缓慢失活”特性，以及高度正调节步骤和Ca ^ 2 激动剂的相互作用。结果表明_1亚基蛋白的分子内结构变化和/或相互作用发挥着核心作用。此外，我们使用多个开放状态模型的计算机计算重建了在克隆平滑肌Ca^2通道中观察到的缓慢失活和U形失活特性。综上所述，L型Ca2通道_1亚基的构象可以以电压依赖性方式从O1态转变为O2态。这种转换不是根据 Hodgkin & Huxley 描述的门控模型预测的。我们想建议对这个基本模型进行一些修改可能是描述更一般条件下的门控动力学所必需的。较少的

It is known that some smooth muscles possess both low- (LVA) and high voltage-activated (HVA) Ca^<2+> channel currents, and that the HVA current component is predominant in all smooth muscles. Previously, we have suggested that the conversion of the Ca^<2+> channel conformation from normal open (O1) to a second open state (O2) during large depoalrization is distinct from the long channel opening induced by Ca^<2+> channel agonists, and that these two mechanisms operate separately. As a result, a combination of DHP Ca^<2+> channel agonists and depolarization produces at least four open states in native smooth muscle Ca^<2+> channels.During the tenure of the present research project entitled 'Molecular Basis of Multiple Open States in Smooth Muscle Calcium Channels and Related Intracellular Signalling', we have first examined whether the multiple open state model can systematically account for the characteristic features of 'U-shaped inactivation' and 'slow deactivation' in CHO cells exp … More ressing only smooth muscle _1 subunit of L-type Ca^<2+> channel (Ca,1.2b), using patch clamp techniques. The experiments have revealed that smooth muscle _1 subunit alone can reproduce 'U-shaped inactivation' and 'slow deactivation' properties, and also interaction of highly positive conditioning steps and Ca^<2+> agonists. The results imply that intramolecular structural changes and/or interactions in _1 subunit protein play the central roles. Furthermore, we have reconstructed the slow deactivation and U-shaped inactivation properties seen in cloned smooth muscle Ca^<2+> channels using computer calculation with multiple open state models. Taken Together, it is concluded that that the conformation of _1 subunit of L-type Ca2+ channel can be converted from O1 to O2 state in a voltage-dependent manner. This conversion is not predicted from the gating model described by Hodgkin & Huxley. We would like to propose that some modification in this basic model is presumably necessary to describe gating kinetics under more general conditions. Less