Probing the role of negatively charged amino acid residues in ion permeation of skeletal muscle ryanodine receptor.
Probing the role of negatively charged amino acid residues in ion permeation of skeletal muscle ryanodine receptor.
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DOI：
10.1529/biophysj.104.056002
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发表时间：
2005-07
影响因子：
3.4
通讯作者：
Ying Wang;Le Xu;D. Pasek;D. Gillespie;G. Meissner
中科院分区：
文献类型：
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作者：
Ying Wang;Le Xu;D. Pasek;D. Gillespie;G. Meissner
序列比较表明，兰尼碱受体（RyRs）具有类似于细菌K+通道KcsA的孔结构。连接RyR中两个最C-末端跨膜段的内腔环具有预测的孔螺旋和与通过X射线分析鉴定的KcsA的选择性过滤器（TVGYG）相似的氨基酸基序（GGGIG）。RyR在连接GGGIG基序和预测的孔螺旋与两个最C-末端跨膜片段的两个区域中具有许多带负电荷的氨基酸残基。我们通过产生单位点突变体来测试这些残基的作用，重点是哺乳动物RyRs中保守的氨基酸残基。骨骼肌Ryanodine受体（RyR 1-D4899和-E4900）中的GGGIG基序后立即用天冬酰胺和谷氨酰胺替换两个酸性残基深刻影响离子渗透和选择性。通过比较，在推定的接头区域中的天冬氨酸和谷氨酸残基的诱变显示出与K+（P（Ca）/P（K））相比接近野生型的K+电导率和对Ca 2+的选择性。结果表明，D4899和E4900侧链上带负电荷的羧基氧是决定RyR离子电导和选择性的主要因素。
Sequence comparison suggests that the ryanodine receptors (RyRs) have pore architecture similar to that of the bacterial K+ channel KcsA. The lumenal loop linking the two most C-terminal transmembrane spanning segments in the RyRs has a predicted pore helix and an amino acid motif (GGGIG) similar to the selectivity filter (TVGYG) of KcsA identified by x-ray analysis. The RyRs have many negatively charged amino acid residues in the two regions linking the GGGIG motif and predicted pore helix with the two most C-terminal transmembrane spanning segments. We tested the role of these residues by generating single-site mutants, focusing on amino acid residues conserved among the mammalian RyRs. Replacement of two acidic residues immediately after the GGGIG motif in skeletal muscle ryanodine receptor (RyR1-D4899 and -E4900) with asparagine and glutamine profoundly affected ion permeation and selectivity. By comparison, mutagenesis of aspartate and glutamate residues in the putative linker regions showed a K+ conductance and selectivity for Ca2+ compared to K+ (P(Ca)/P(K)) close to wild-type. The results show that the negatively charged carboxyl oxygens of D4899 and E4900 side chains are major determinants of RyR ion conductance and selectivity.