tailieunhanh - Báo cáo khoa học: Functional importance of charged residues within the putative intracellular loops in pH regulation by Na+ ⁄ H+ exchanger NHE1

The plasma membrane Na + ⁄H + exchanger 1 is activated in response to various extrinsic factors, and this process is regulated by an intracellular pH-sensing mechanism. To identify the candidate residues responsible for intracellular pH regulation, we analyzed the functional properties of engi-neered Na + ⁄H + exchanger 1 mutants with charge-reversal mutations of charged residues located in the intracellular loops. | ễFEBS Journal Functional importance of charged residues within the putative intracellular loops in pH regulation by Na H exchanger NHE1 Takashi Hisamitsu Keiji Yamada Tomoe Y. Nakamura and Shigeo Wakabayashi Department of Molecular Physiology NationalCardiovascular Center Research Institute Suita Japan Keywords charge-reversalmutation Na H exchanger pH regulation surface labeling transport kinetics Correspondence S. Wakabayashi Department of Molecular Physiology NationalCardiovascular Center Research Institute Suita Osaka 565-8565 Japan Fax 81 6 6835 5314 Tel 81 6 6833 5012 E-mail wak@ These authors contributed equally to this work Received 13 February 2007 revised 21 June 2007 accepted 28 June 2007 doi The plasma membrane Na H exchanger 1 is activated in response to various extrinsic factors and this process is regulated by an intracellular pH-sensing mechanism. To identify the candidate residues responsible for intracellular pH regulation we analyzed the functional properties of engineered Na H exchanger 1 mutants with charge-reversal mutations of charged residues located in the intracellular loops. Na H exchanger 1 mutants with mutations at 11 positions were well expressed in the plasma membrane but that with E247R was not suggesting that Glu247 is important for the functional expression of Na H exchanger 1. Charge-reversal mutations of Glu131 E131R E131K and Arg327 R327E resulted in a shift in the intracellular pH dependence of the exchange activity measured by 22Na uptake to the acidic side and it abolished the response to growth factors and a hyperosmotic medium however mutations of Asp448 D448R and Arg500 R500E slightly shifted it to the alkaline side. In E131R in addition to the change in intracellular pH dependence the affinities for extracellular Na Li and the inhibitor 5- N-ethyl-N-iso-propyl amiloride significantly increased. Furthermore charge-conserved mutation of E131 E131D was found to have no effect .