Erickson Michael G, Liang Haoya, Mori Masayuki X, Yue David T
The Johns Hopkins University School of Medicine, Department of Biomedical Engineering, Calcium Signals Laboratory, Baltimore, MD 21205, USA.
Neuron. 2003 Jul 3;39(1):97-107. doi: 10.1016/s0896-6273(03)00395-7.
L-type Ca(2+) channels possess a Ca(2+)-dependent inactivation (CDI) mechanism, affording feedback in diverse neurobiological settings and serving as prototype for unconventional calmodulin (CaM) regulation emerging in many Ca(2+) channels. Crucial to such regulation is the preassociation of Ca(2+)-free CaM (apoCaM) to channels, facilitating rapid triggering of CDI as Ca(2+)/CaM shifts to a channel IQ site (IQ). Progress has been hindered by controversy over the preassociation site, as identified by in vitro assays. Most critical has been the failure to resolve a functional signature of preassociation. Here, we deploy novel FRET assays in live cells to identify a 73 aa channel segment, containing IQ, as the critical preassociation pocket. IQ mutations disrupting preassociation revealed accelerated voltage-dependent inactivation (VDI) as the functional hallmark of channels lacking preassociated CaM. Hence, the alpha(1C) IQ segment is multifunctional-serving as ligand for preassociation and as Ca(2+)/CaM effector site for CDI.
L型钙通道具有钙依赖性失活（CDI）机制，在多种神经生物学环境中提供反馈，并作为许多钙通道中出现的非常规钙调蛋白（CaM）调节的原型。这种调节的关键是无钙CaM（脱钙钙调蛋白，apoCaM）与通道的预结合，随着Ca2+/CaM转移到通道IQ位点（IQ），促进CDI的快速触发。体外试验确定的预结合位点存在争议，这阻碍了研究进展。最关键的是未能解析预结合的功能特征。在这里，我们在活细胞中采用新型荧光共振能量转移（FRET）试验，以确定一个包含IQ的73个氨基酸的通道片段作为关键的预结合口袋。破坏预结合的IQ突变显示，加速电压依赖性失活（VDI）是缺乏预结合CaM的通道的功能标志。因此，α1C IQ片段具有多种功能——作为预结合的配体和作为CDI的Ca2+/CaM效应位点。
