Molecular Dissection of Calmodulin Domain Functions

描述(申请人提供)：钙调节离子通道控制神经和肌肉的基本过程。与电压依赖性钠通道家族相关的通道病变(记为NaV1.n，VDSC或VGSC)包括几种严重形式的癫痫、心脏长QT综合征3和室颤、家族性自闭症、疼痛不敏感，以及动作电位产生和传播的其他缺陷。大多数NAV家族的成员通过钙调素(CaM)对钙作出反应，CaM是真核生物中一种重要的钙传感器，它由两个高度同源的结构域(N和C)组成，由一个调节连接子连接。新的溶液结构显示了apo(无钙)C如何与成孔细胞内C末端尾部的IQ基序(IQxxx[R，K]Gxxx[R，K])结合 NaV1.2(2KXW)和NaV1.5(2L53)的α亚基。对于两者来说，钙离子与CaM的结合改变了C的构象，降低了它对NAV IQ基序的亲和力。虽然这些结构和热力学研究表明N不与NAV智商基序结合，但遗传研究表明N的突变影响NAV功能。因此，在我们对这一钙介导的转换过程的理解中，一个显著的差距是N如何以及在哪里与NAV相关联。我们假设CaM作为钙离子触发的通道组织者；CaM的N和C结构域将NAV的不同部分聚集在一起，调节构象和电导。我们认为载脂蛋白Cam在智商基序上被C拴住。当钙离子与CaM结合时，N结合到它在NAV中的首选位置，而C要么(A)以相同的取向在IQ基序上打开并扭曲，要么(B)以相反的取向释放并重新结合。这些取向将以不同的方式限制N可用的NAV接口。为了确定CaM如何将细胞内[Ca~(2+)]的变化转化为构象功，并调节NAV的多种异构体，我们将通过测试N和C如何识别自然发生的人类NAV异构体的变化，以及NAV的致病突变如何独特地影响apo与钙饱和的N和C的结合，来确定N和C的不同角色。我们提出了三个目标，重点是中枢神经系统(CNS)中发现的钠通道。第一个目标将确定载脂蛋白和钙饱和的CaM C结构域如何与NAV的致病突变IQ基序结合。第二个目的将确定CaM对NAV智商基序和NAV“EF-Hands”之间相互作用的影响。第三个目的将确定载脂蛋白和(钙)2-CaM N-结构域与NAV的异构体结合的位置。CaM的结构将通过结晶学、核磁共振和CD来研究，而动力学和能量滴定将通过编码已知和假定的CaM结合结构域的荧光生物传感器、核磁共振和稳态荧光监测的钙滴定来确定。我们将发现CaM和NAV之间的关键大分子取向，提供包含EF-Hand和IQ基序的NAV尾部片段的第一结构，并表明CaM介导的多个与癫痫相关的疾病相关突变，或被确定为自闭症谱系障碍的候选基因。

DESCRIPTION (provided by applicant): Calcium-regulated ion channels control essential processes in nerves and muscles. Channelopathies associated with the voltage-dependent sodium channel family (denoted NaV1.n, VDSC or VGSC) include several severe forms of epilepsy, cardiac "Long QT" syndrome 3 and ventricular fibrillation, familial autism, pain insensitivity, and other defects in the generation and propagation of action potentials. Most members of the NaV family of channels respond to calcium via regulation by calmodulin (CaM), an essential eukaryotic calcium sensor that is comprised of two highly homologous domains (N and C) connected by a regulatory linker. New solution structures show how apo (calcium-free) C binds to an IQ motif (IQxxx[R,K]Gxxx[R,K]) in the intracellular C-terminal tail of the pore-forming alpha subunit of NaV1.2 (2KXW) and NaV1.5 (2L53). For both, Ca2+ binding to CaM changes the conformation of C and lowers its affinity for the NaV IQ motif. Although these structures and thermodynamic studies show that N does not bind to the NaV IQ motif, genetic studies indicate that mutations in N affect NaV function. Thus, a significant gap in our understanding of this calcium- mediated switching process is how and where N associates with NaV. We hypothesize that CaM serves as a calcium-triggered channel organizer; the N and C domains of CaM bring together distinct parts of NaV to regulate conformation and conductivity. We propose that apo CaM is tethered by C at the IQ motif. When Ca2+ binds to CaM, N binds to its preferred site in NaV, while C either (a) opens and twists on the IQ motif, with the same orientation, or (b) releases and rebinds with opposite orientation. These orientations would restrict the NaV interfaces available to N in different ways. To determine how CaM translates changes in intracellular [Ca2+] into conformational work, and regulates multiple isoforms of NaV, we will determine the distinct roles of N and C by testing how they recognize naturally occurring variations in isoforms of human NaV and how disease-causing mutations in NaV uniquely affect binding of apo and calcium-saturated N and C. We propose three Aims focused on sodium channels found in the central nervous system (CNS). The first Aim will determine how apo and Ca2+-saturated CaM C-domain binds disease-causing mutant IQ motifs of NaV. The second Aim will determine the effect of CaM on interactions between NaV IQ motifs and NaV "EF-Hands". The third Aim will determine where apo and (Ca2+)2-CaM N-domain are binding to isoforms of NaV. Structures of CaM will be studied by crystallography, NMR, and CD, while dynamics and energetics from titrations will be determined with fluorescent biosensors encoding known and putative CaM-binding domains, NMR, and calcium titrations monitored by steady-state fluorescence. We will discover key macromolecular orientations between CaM and NaV, provide the first structure of an NaV tail fragment containing both the EF-hand and IQ motif, and indicate the CaM-mediated contribution to multiple disease-associated mutations related to epilepsy, or identified as candidates for autism-spectrum disorders.