Identification of Cytoplasmic Subdomains That Control pH-Sensing of the Na+/H+ Exchanger (NHE1): pH-Maintenance, ATP-Sensitive, and Flexible Loop Domains.

摘要: To precisely identify the cytoplasmic subdomains that are responsible for intracellular pH (pHi)-sensitivity, ATP depletion-induced inhibition and Ca2+ activation of Na+/H+ exchanger (NHE1), we generated a set deletion mutants carboxyl-terminated domain expressed them in exchanger-deficient cell line PS120. We evaluated pHi-sensitivity these by measuring resting pHi cells placed an acidic medium (pH 6.0) pHi-dependence 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake. Detailed analysis revealed NHE1 is consists at least four terms unstimulated NHE1: I, aa 516-590/595; II, 596-635; III 636-659; IV, 660-815. Subdomains II IV were silent pHi-sensitivity. Subdomain I had pHi-maintenance function, preserving physiological range, whereas subdomain III, overlapping with high affinity calmodulin (CaM)-binding site, exhibited autoinhibitory function. Deletion abolished decrease induced depletion, indicating plays crucial role this phenomenon. rendered depletion less efficient, suggesting possible interaction between III. On other hand, tandem elongation insertion did not affect either inhibitory function or removal ionomycin thrombin. However, partially effect thus seems to as mobile "flexible loop," permitting CaM-binding exert its normal These findings, together our previous data, support concept ATP, Ca2+, growth factors regulate via mechanism involving direct indirect interactions specific "H(+)-modifier site.".