Molecular Dissection of Calmodulin Domain Functions

描述（由申请人提供）：钙调节离子通道控制神经和肌肉中的基本过程。与电压依赖性钠通道家族（表示为NaV1.n、VDSC或VGSC）相关的癫痫病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、疼痛不敏感性以及动作电位产生和传播中的其他缺陷。NaV通道家族的大多数成员通过钙调蛋白（CaM）的调节对钙做出反应，钙调蛋白是一种必需的真核钙传感器，由两个高度同源的结构域（N和C）组成，通过调节接头连接。新的溶液结构显示了载脂蛋白（无钙）C如何与成孔蛋白的细胞内C末端尾部的IQ基序（IQxxx[R，K]Gxxx[R，K]）结合。 NaV1.2（2KXW）和NaV1.5（2L 53）的α亚基。对于这两种情况，Ca 2+与CaM的结合改变了C的构象，并降低了其对NaV IQ基序的亲和力。尽管这些结构和热力学研究表明N不与NaV IQ基序结合，但遗传学研究表明N的突变会影响NaV功能。因此，我们对这种钙介导的转换过程的理解中的一个显著差距是N如何以及在何处与NaV缔合。我们假设，钙调素作为钙触发的通道组织者，钙调素的N和C域汇集不同的部分NaV调节构象和电导率。我们建议，载脂蛋白钙调素是由C在智商基序。当Ca 2+与CaM结合时，N结合到NaV中的其优选位点，而C（a）以相同的方向在IQ基序上打开并扭曲，或（B）以相反的方向释放并重新结合。这些方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 +]的变化转化为构象工作，并调节NaV的多种亚型，我们将通过测试N和C如何识别人类NaV亚型中天然存在的变异以及NaV中的致病突变如何独特地影响apo和钙饱和N和C的结合来确定N和C的不同作用。我们提出了三个目标集中在钠通道发现在中枢神经系统（CNS）。第一个目标将确定载脂蛋白和Ca 2+饱和的CaM C结构域如何结合NaV的致病突变体IQ基序。第二个目的将确定CaM对NaV IQ基序和NaV“EF-手”之间相互作用的影响。第三个目标将确定apo和（Ca 2+）2-CaM N-结构域与NaV同种型结合的位置。钙调素的结构将通过晶体学，NMR和CD进行研究，而滴定的动力学和能量学将通过编码已知和推定的钙调素结合结构域的荧光生物传感器，NMR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-手和IQ基序的NaV尾片段的第一个结构，并表明CaM介导的与癫痫相关的多种疾病相关突变的贡献，或被确定为自闭症谱系障碍的候选者。 公共卫生相关性：NaV家族中的人电压门控钠通道由钙调蛋白（CaM）调节，并负责中枢神经系统、心脏和骨骼肌中动作电位的上升相。已知影响钙反应、折叠、温度敏感性和蛋白质总长度的突变会导致癫痫、心脏缺陷，并且是自闭症和ALS（“Lou Gehrig's Disease”）潜在基因的候选者。虽然新生携带者可能没有后代，高通量筛选开辟了新的机会，以确定有害的序列变化的钙调素结合区域。我们将确定钙调素如何重塑细胞内的C-末端尾巴的NaV提供钙介导的开关。发现钙调素每个域的调节作用为开发未来的治疗奠定了基础，并将提供有关癫痫和其他神经系统疾病药物副作用的信息。

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. PUBLIC HEALTH RELEVANCE: Human voltage-gated sodium channels in the NaV family are regulated by calmodulin (CaM) and responsible for the rising phase of action potentials in the central nervous system, heart and skeletal muscle. Mutations that affect calcium response, folding, temperature sensitivity and the overall length of the protein are known to cause epilepsy, cardiac defects, and are candidates for genes underlying autism and ALS ("Lou Gehrig's Disease"). Although de novo carriers may not have offspring, high-throughput screening has opened new opportunities to identify deleterious sequence changes in the CaM-binding regions. We will determine how CaM remodels the intracellular C-terminal tail of NaV to provide calcium-mediated switching. Discovering the regulatory role of each domain of CaM lays the groundwork for developing future therapies and will provide information regarding side-effects of medications taken for epilepsy and other neurological disorders.