Lee Brian L, Liu Yongsheng, Li Xiuju, Sykes Brian D, Fliegel Larry
Department of Biochemistry, University of Alberta, Alberta, Canada.
Biochim Biophys Acta. 2012 Nov;1818(11):2783-90. doi: 10.1016/j.bbamem.2012.06.021. Epub 2012 Jul 6.
The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein. It regulates intracellular pH by removing a single intracellular H(+) in exchange for one extracellular Na(+). The membrane domain of NHE1 comprises the 500 N-terminal amino acids and is made of 12 transmembrane segments. The extracellular loops of the transmembrane segments are thought to be involved in cation coordination and inhibitor sensitivity. We have characterized the structure and function of amino acids 278-291 representing extracellular loop 4. When mutated to Cys, residues F277, F280, N282 and E284 of EL4 were sensitive to mutation and reaction with MTSET inhibiting NHE1 activity. In addition they were found to be accessible to extracellular applied MTSET. A peptide of the amino acids of EL4 was mostly unstructured suggesting that it does not provide a rigid structured link between TM VII and TM VIII. Our results suggest that EL4 makes an extension upward from TM VII to make up part of the mouth of the NHE1 protein and is involved in cation selectivity or coordination. EL4 provides a flexible link to TM VIII which may either allow movement of TM VII or allow TM VIII to not be adjacent to TM VII.
哺乳动物钠氢交换体同工型1（NHE1）是一种广泛表达的质膜蛋白。它通过将一个细胞内氢离子与一个细胞外钠离子交换来调节细胞内pH值。NHE1的膜结构域由500个N端氨基酸组成，由12个跨膜片段构成。跨膜片段的细胞外环被认为参与阳离子配位和抑制剂敏感性。我们已经对代表细胞外环4的氨基酸278 - 291的结构和功能进行了表征。当突变为半胱氨酸时，EL4的F277、F280、N282和E284残基对突变以及与抑制NHE1活性的MTSET反应敏感。此外，发现它们可被细胞外施加的MTSET接触。EL4氨基酸的肽大多是非结构化的，这表明它在跨膜结构域VII和VIII之间不提供刚性的结构化连接。我们的结果表明，EL4从跨膜结构域VII向上延伸，构成NHE1蛋白口部的一部分，并参与阳离子选择性或配位。EL4为跨膜结构域VIII提供了一个灵活的连接，这可能允许跨膜结构域VII移动，或者允许跨膜结构域VIII不与跨膜结构域VII相邻。
