Solution NMR Structure of the C-terminal EF-hand Domain of Human Cardiac Sodium Channel NaV1.5
Solution NMR Structure of the C-terminal EF-hand Domain of Human Cardiac Sodium Channel NaV1.5
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DOI：
10.1074/jbc.m807747200
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发表时间：
2009-03-06
影响因子：
4.8
通讯作者：
Chazin, Walter J.
中科院分区：
文献类型：
--
作者：
Chagot, Benjamin;Potet, Franck;Chazin, Walter J.
电压门控钠通道Na(V)1.5负责心脏组织中动作电位的初始上升。细胞内钙水平调节Na(V)1.5的失活门控，部分通过c端EF-hand钙结合域。这种结构的重要性被强调的事实是，在这个区域内的突变与特定的心律失常综合征有关。为了阐明钙调控通道功能的分子基础，我们利用多维异核磁共振确定了c端EF-hand结构域的溶液结构。该结构证实了EF-hand结构域蛋白共有的四螺旋束的存在。然而，该结构域的位置相对于基于序列中普遍存在的12个残基EF-hand钙结合环所预测的位置发生了移位。这一发现与报道的分离的EF-hand结构域的弱钙亲和力一致；高亲和结合仅在EF-hand结构域c端有额外60个残基的结构体中观察到，包括钙调节装置的核心IQ基序。用等温滴定量热法和核磁共振波谱法对IQ基序肽与EF-hand结构域的结合进行了表征。肽结合在EF-hand结构域的螺旋I和IV之间，类似于靶肽与其他EF-hand钙结合蛋白的结合。这些结果表明Na(V)1.5钙传感装置的内在（EF-hand结构域）和外在（钙调素）组分耦合的分子基础。
The voltage-gated sodium channel Na(V)1.5 is responsible for the initial upstroke of the action potential in cardiac tissue. Levels of intracellular calcium modulate inactivation gating of Na(V)1.5, in part through a C-terminal EF-hand calcium binding domain. The significance of this structure is underscored by the fact that mutations within this domain are associated with specific cardiac arrhythmia syndromes. In an effort to elucidate the molecular basis for calcium regulation of channel function, we have determined the solution structure of the C-terminal EF-hand domain using multidimensional heteronuclear NMR. The structure confirms the existence of the four-helix bundle common to EF-hand domain proteins. However, the location of this domain is shifted with respect to that predicted on the basis of a consensus 12-residue EF-hand calcium binding loop in the sequence. This finding is consistent with the weak calcium affinity reported for the isolated EF-hand domain; high affinity binding is observed only in a construct with an additional 60 residues C-terminal to the EF-hand domain, including the IQ motif that is central to the calcium regulatory apparatus. The binding of an IQ motif peptide to the EF-hand domain was characterized by isothermal titration calorimetry and nuclear magnetic resonance spectroscopy. The peptide binds between helices I and IV in the EF-hand domain, similar to the binding of target peptides to other EF-hand calcium-binding proteins. These results suggest a molecular basis for the coupling of the intrinsic (EF-hand domain) and extrinsic (calmodulin) components of the calcium-sensing apparatus of Na(V)1.5.