Mutational analysis of the α-1 repeat of the cardiac Na+-Ca2+ exchanger
Mutational analysis of the α-1 repeat of the cardiac Na+-Ca2+ exchanger
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DOI：
10.1074/jbc.m411899200
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发表时间：
2005-01-14
影响因子：
4.8
通讯作者：
Philipson, KD
中科院分区：
文献类型：
--
作者：
Ottolia, M;Nicoll, DA;Philipson, KD
Na+-Ca2+交换器包含序列同源的内部区域，称为α重复序列。第一个区域（α -1重复）包括跨膜节段（tms） 2和3的部分和一个连接体模型为可重入环。为了确定α -1重复序列的重入环和tms3部分在交换功能中的作用，我们生成了一系列突变体，并检查了离子结合、运输和调控特性。虽然Na+的Hill系数和Na+依赖性失活速率降低，但可重入环的突变并未实质性地改变交换剂的输运性质。tms3突变对交换剂活性的影响更为显著。在10个位点的突变中，有3个表现为野生型交换器（V137C、A141C、M144C）。其他两个位置的突变体不表达活性（Ser(139)）或表达活性非常低（Gly(138)）。在143号位置产生了6个不同的突变；只有N143D有活性，且表现出野生型特征。在这个位置对天冬酰胺或天冬氨酸残基的高度特异性需求可能表明Asn（143）在运输机制中起关键作用。Ala（140）和Ile（147）的突变降低了细胞内Na+的亲和力，而Phe（145）的突变增加了Na+的亲和力。Na+结合的协同性也发生了改变。在任何情况下Ca2+亲和力都没有改变。tms3可能形成结合Na+但不结合Ca2+的位点的一部分。我们得出结论，tms3参与Na+结合和转运，但之前提出的重入环的作用需要重新评估。
The Na+-Ca2+ exchanger contains internal regions of sequence homology known as the alpha repeats. The first region (alpha-1 repeat) includes parts of transmembrane segments (TMSs) 2 and 3 and a linker modeled to be a reentrant loop. To determine the involvement of the reentrant loop and TMS 3 portions of the alpha-1 repeat in exchanger function, we generated a series of mutants and examined ion binding and transport and regulatory properties. Mutations in the reentrant loop did not substantially modify transport properties of the exchanger though the Hill coefficient for Na+ and the rate of Na+-dependent inactivation were decreased. Mutations in TMS 3 had more striking effects on exchanger activity. Of mutations at 10 positions, 3 behaved like the wild-type exchanger (V137C, A141C, M144C). Mutants at two other positions expressed no activity (Ser(139)) or very low activity (Gly(138)). Six different mutations were made at position 143; only N143D was active, and it displayed wild-type characteristics. The highly specific requirement for an asparagine or aspartate residue at this position may indicate a key role for Asn(143) in the transport mechanism. Mutations at residues Ala(140) and Ile(147) decreased affinity for intracellular Na+, whereas mutations at Phe(145) increased Na+ affinity. The cooperativity of Na+ binding was also altered. In no case was Ca2+ affinity changed. TMS 3 may form part of a site that binds Na+ but not Ca2+. We conclude that TMS 3 is involved in Na+ binding and transport, but previously proposed roles for the reentrant loop need to be reevaluated.