IDENTIFICATION OF CYTOPLASMIC SUBDOMAINS THAT CONTROL PH-SENSING OF THE NA+ H+ EXCHANGER (NHE1) - PH-MAINTENANCE, ATP-SENSITIVE, AND FLEXIBLE LOOP DOMAINS/

To precisely identify the cytoplasmic subdomains that are responsible for the intracellular pH (pH(i))-sensitivity, ATP depletion-induced in hibition and Ca2+ activation of the Na+/H+ exchanger (NHE1), we genera ted a set of deletion mutants of carboxyl-terminal cytoplasmic domain and expressed them in the exchanger-deficient cell line PS120. We eval uated pH(i)-sensitivity of these mutants by measuring the resting pH(i ) in cells placed in an acidic medium (pH 6.0) and pH(i)-dependence of 5-(N-ethyl-N-isopropyl)amiloride-sensitive Na-22(+) uptake. Detailed analysis revealed that the cytoplasmic domain of NHE1 is consists of a t least four subdomains in terms of pH(i)-sensitivity of the unstimula ted NHE1:I, aa 516-590/595; II, aa 596-635; III, aa 636-659; and IV. a a 660-815. Subdomains II and IV were silent for pH(i)-sensitivity. Sub domain I had a pH(i)-maintenance function, preserving pH(i)-sensitivit y in a physiological range, whereas subdomain III, overlapping with th e high affinity calmodulin (CaM)-binding site, exhibited an autoinhibi tory function. Deletion of subdomain I abolished the decrease of pH(i) -sensitivity induced by cell ATP depletion, indicating that domain I p lays a crucial role in this phenomenon. Deletion of subdomain III rend ered the inhibition by ATP depletion less efficient, suggesting the po ssible interaction between subdomains I and III. On the other hand, ta ndem elongation of subdomain II by insertion did not affect either the inhibitory function of domain III or the removal of this inhibition b y ionomycin or thrombin. However, deletion of subdomain II partially a bolished the inhibitory effect of subdomain III. Subdomain II thus see ms to function as a mobile ''flexible loop,'' permitting the CaM-bindi ng subdomain III to exert its normal function. These findings, togethe r with our previous data, support a concept that cell ATP, Ca2+, and g rowth factors regulate NHE1 via a mechanism involving direct or indire ct interactions of specific cytoplasmic subdomains with the ''H+-modif ier site.''