Dulhunty Angela F, Curtis Suzanne M, Cengia Louise, Sakowska Magdalena, Casarotto Marco G
John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia.
Biochem J. 2004 Apr 1;379(Pt 1):161-72. doi: 10.1042/BJ20031096.
We show that peptide fragments of the dihydropyridine receptor II-III loop alter cardiac RyR (ryanodine receptor) channel activity in a cytoplasmic Ca2+-dependent manner. The peptides were AC (Thr-793-Ala-812 of the cardiac dihydropyridine receptor), AS (Thr-671-Leu-690 of the skeletal dihydropyridine receptor), and a modified AS peptide [AS(D-R18)], with an extended helical structure. The peptides added to the cytoplasmic side of channels in lipid bilayers at > or = 10 nM activated channels when the cytoplasmic [Ca2+] was 100 nM, but either inhibited or did not affect channel activity when the cytoplasmic [Ca2+] was 10 or 100 microM. Both activation and inhibition were independent of bilayer potential. Activation by AS, but not by AC or AS(D-R18), was reduced at peptide concentrations >1 mM in a voltage-dependent manner (at +40 mV). In control experiments, channels were not activated by the scrambled AS sequence (ASS) or skeletal II-III loop peptide (NB). Resting Ca2+ release from cardiac sarcoplasmic reticulum was not altered by peptide AC, but Ca2+-induced Ca2+ release was depressed. Resting and Ca2+-induced Ca2+ release were enhanced by both the native and modified AS peptides. NMR revealed (i) that the structure of peptide AS(D-R18) is not influenced by [Ca2+] and (ii) that peptide AC adopts a helical structure, particularly in the region containing positively charged residues. This is the first report of specific functional interactions between dihydropyridine receptor A region peptides and cardiac RyR ion channels in lipid bilayers.
我们发现,二氢吡啶受体II-III环的肽片段以细胞质Ca2+依赖的方式改变心脏兰尼碱受体(RyR)通道活性。这些肽分别是AC(心脏二氢吡啶受体的Thr-793-Ala-812)、AS(骨骼肌二氢吡啶受体的Thr-671-Leu-690)以及具有延伸螺旋结构的修饰AS肽[AS(D-R18)]。当细胞质[Ca2+]为100 nM时,以≥10 nM添加到脂质双层中通道细胞质侧的这些肽可激活通道,但当细胞质[Ca2+]为10或100 μM时,它们要么抑制通道活性,要么不影响通道活性。激活和抑制均与双层电位无关。在肽浓度>1 mM时,AS引起的激活呈电压依赖性降低(在+40 mV时),而AC或AS(D-R18)则不会。在对照实验中,通道不会被乱序的AS序列(ASS)或骨骼肌II-III环肽(NB)激活。肽AC不会改变心脏肌浆网的静息Ca2+释放,但会抑制Ca2+诱导的Ca2+释放。天然和修饰的AS肽均可增强静息和Ca2+诱导的Ca2+释放。核磁共振显示:(i)肽AS(D-R18)的结构不受[Ca2+]影响;(ii)肽AC呈现螺旋结构,尤其是在含有带正电荷残基的区域。这是关于脂质双层中二氢吡啶受体A区肽与心脏RyR离子通道之间特异性功能相互作用的首次报道。