Structural and functional analysis of extracellular loop 4 of the Nhe1 isoform of the Na(+)/H(+) exchanger

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.