Peptide ligands of the cardiac ryanodine receptor as super-resolution imaging probes
Peptide ligands of the cardiac ryanodine receptor as super-resolution imaging probes
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DOI：
10.1101/2020.06.26.131854
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发表时间：
2020-06
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通讯作者：
T. M. Sheard;Luke A. Howlett;Hannah M. Kirton;Zhaokang Yang;G. Gurrola;D. Steele;I. Jayasinghe;H. Valdivia;J. Colyer
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作者：
T. M. Sheard;Luke A. Howlett;Hannah M. Kirton;Zhaokang Yang;G. Gurrola;D. Steele;I. Jayasinghe;H. Valdivia;J. Colyer
为了研究心脏病理性重构和钙通道功能状态改变的结构基础，我们试图将心脏钙通道的高亲和力配体ryanodine受体（RyR2）重新用于超分辨率成像探针。在荧光成像实验中使用了Imperacalcin（IpCa）（一种诱导通道亚传导状态的蝎毒素肽）和DPc10（一种对应于RyR2序列的合成肽，其复制致炎性CPVT功能变化）。将分离的成年大鼠心室心肌细胞用皂苷透化并与每种肽孵育。IpCa-A546被隔离到线粒体中。这是通过用离子载体FCCP处理透化细胞来防止的，在共聚焦成像中揭示了具有与RyR2簇弱共定位的条纹染色模式。用扩增显微镜（proExM）（~70 nm）在更高分辨率下证实了差的特异性（作为RyR2成像探针）。DPc10-FITC标记的条纹图案，在共聚焦和proExM中与RyR2簇标记具有中度共定位。还存在Z盘、闰盘和细胞核的广泛非靶标记，其不受孵育时间或10 mM咖啡因的影响。无活性肽mut-DPc10-FITC（不引起功能效应）显示出类似的标记模式。通过两种肽缀合物显著标记与RyR2无关的结构，使得它们在活的分离的心肌细胞中作为RyR2的高度特异性成像探针的用途极具挑战性。我们研究了RyR2结构内的天然DPc10序列，以了解结构域相互作用和提出的肽结合机制。天然DPc10序列不直接与另一个结构域相互作用，而是位于一个这样的结构域界面的下游。天然序列中的兔Arg2475（相当于人Arg2474，在CPVT中突变）是最易接近的部分，也是肽干扰的最可能位置，表明FITC放置不影响肽结合。
To study the structural basis of pathological remodelling and altered calcium channel functional states in the heart, we sought to re-purpose high-affinity ligands of the cardiac calcium channel, the ryanodine receptor (RyR2), into super-resolution imaging probes. Imperacalcin (IpCa), a scorpion toxin peptide which induces channel sub-conduction states, and DPc10, a synthetic peptide corresponding to a sequence of the RyR2, which replicates arrhythmogenic CPVT functional changes, were used in fluorescent imaging experiments. Isolated adult rat ventricular cardiomyocytes were saponin-permeabilised and incubated with each peptide. IpCa-A546 became sequestered into the mitochondria. This was prevented by treatment of the permeabilised cells with the ionophore FCCP, revealing a striated staining pattern in confocal imaging which had weak colocalisation with RyR2 clusters. Poor specificity (as an RyR2 imaging probe) was confirmed at higher resolution with expansion microscopy (proExM) (~70 nm). DPc10-FITC labelled a striated pattern, which had moderate colocalisation with RyR2 cluster labelling in confocal and proExM. There was also widespread non-target labelling of the Z-discs, intercalated discs, and nuclei, which was unaffected by incubation times or 10 mM caffeine. The inactive peptide mut-DPc10-FITC (which causes no functional effects) displayed a similar labelling pattern. Significant labelling of structures unrelated to RyR2 by both peptide conjugates makes their use as highly specific imaging probes of RyR2 in living isolated cardiomyocytes highly challenging. We investigated the native DPc10 sequence within the RyR2 structure to understand the domain interactions and proposed mechanism of peptide binding. The native DPc10 sequence does not directly interact with another domain, and but is downstream of one such domain interface. The rabbit Arg2475 (equivalent to human Arg2474, mutated in CPVT) in the native sequence is the most accessible portion and most likely location for peptide disturbance, suggesting FITC placement does not impact peptide binding.