Functional consequences of mutations of conserved, polar amino acids in transmembrane sequences of the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum
Functional consequences of mutations of conserved, polar amino acids in transmembrane sequences of the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum
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DOI：
10.1074/jbc.273.48.31867
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发表时间：
1998-11-27
影响因子：
4.8
通讯作者：
MacLennan, DH
中科院分区：
文献类型：
--
作者：
Du, GG;MacLennan, DH
通过诱变，研究了兔骨骼肌兰尼碱受体（RyR 1）预测的跨膜序列中高度保守的极性小氨基酸在Ca 2+释放通道功能中的潜在作用，酸性氨基酸Asp（3987），Glu（4032），Asp（4815），Asp（4917），Asp（4938），并且将Asp（4969）和酰胺化残基Asn（4034）Asn（4037）Asn（4574）Asn（4805）、Asn（4806）和Gln（4933）以及Gly（4033）突变为Ala，并且将Ala（3988）突变为瓦尔，当在HEK-293细胞中表达并用咖啡因或4-氯间甲酚攻击时，突变体E4032 A、N4806 A、D4815 A和D4917 A没有响应，表明Ca 2+释放通道功能受损。这些突变体均未表现出[H-3]ryanodine的特异性结合。突变体N4805 A和Q4933 A对咖啡因和4-氯间甲酚的反应减弱，但[H-3]ryanodine结合没有改变。与RyR 1相比，其他突变体和突变体E4032 D、N4806 Q或D、D4815 N或E和D4938 N或E的反应没有改变，然而，突变体E4032 Q、D4917 N或E和Q4933 N或E既不显示咖啡因反应，也不显示4-氯间甲酚反应，也不显示[H-3]ryanodine结合。沉降分析表明，非功能性突变体确实含有四聚体复合物，这意味着跨膜序列中的特定突变不会导致功能性通道组装中的缺陷。这些结果支持氨基酸Glu（4032）（M2）、Asn（4806）（M7）、Asp（4815）（M7）、Asp（4917）（M10）和Gln（4933）（M10）参与通道功能和调节的观点。
The potential role in Ca2+ release channel function of highly conserved, polar, and small amino acids in predicted transmembrane sequences in the rabbit skeletal muscle ryanodine receptor (RyR1) was investigated through mutagenesis, Acidic amino acids Asp(3987), Glu(4032), Asp(4815), Asp(4917), Asp(4938),and Asp(4969) and amidated residues Asn(4034) Asn(4037) Asn(4574) Asn(4805), Asn(4806), and Gln(4933), and Gly(4033) were mutated to Ala, and Ala(3988) was mutated to Val, When expressed in HEK-293 cells and challenged with either caffeine or 4-chloro-m cresol, mutants E4032A, N4806A, D4815A, and D4917A did not respond, indicating that Ca2+ release channel function was impaired. None of these mutants exhibited specific binding of [H-3]ryanodine. Mutants N4805A and Q4933A showed a diminished response to both caffeine and 4-chloro-m-cresol, but [H-3]ryanodine binding was not altered. Other mutant responses and the responses of mutants E4032D, N4806Q or D, D4815N or E, and D4938N or E were unaltered when compared with RyR1, However, mutants E4032Q, D4917N or E, and Q4933N or E displayed neither caffeine nor 4-chloro-m-cresol response nor [H-3]ryanodine binding. Sedimentation assays indicated that the nonfunctional mutants did contain tetrameric complexes, implying that defects in the assembly of a functional channel did not occur with specific mutations in transmembrane sequences. These results support the view that amino acids Glu(4032) (M2), Asn(4806) (M7), Asp(4815) (M7), Asp(4917) (M10), and Gln(4933) (M10) are involved in channel function and regulation.