MUTATION OF CALMODULIN-BINDING SITE RENDERS THE NA+ H+ EXCHANGER (NHE1) HIGHLY H+-SENSITIVE AND CA2+ REGULATION-DEFECTIVE/

The ubiquitous plasma membrane Na+/H+ exchanger (NHE1) is rapidly acti vated in response to various extracellular signals. To understand how the intracellular Ca2+ is involved in this activation process, we inve stigated the effect of Ca2+ ionophore ionomycin on activity of the wil d-type or mutant NHE1 expressed in the exchanger deficient fibroblasts (PS120). In wild-type transfectants, a short (up to 1 min) incubation with ionomycin induced a significant alkaline shift (similar to 0.2 p H unit) in the intracellular pH (pH(i)) dependence of the rate of 5-(N -ethyl-N-isopropyl) amiloride-sensitive Na-22(+) uptake, without chang es in the cell volume and phosphorylation state of NHE1. Mutations tha t prevented calmodulin (CaM) binding to a high affinity binding region (region A, amino acids 636-656) rendered NHE1 constitutively active b y inducing a similar alkaline shift in pH(i) dependence of Na+/H+ exch ange. These same mutations abolished the ionomycin-induced NHE1 activa tion. These data suggest that CaM-binding region A functions as an ''a utoinhibitory domain'' and that Ca2+/CaM activates NHE1 by binding to region A and thus abolishing its inhibitory effect. Furthermore, we fo und that a short stimulation with thrombin and ionomycin had apparentl y no additive effects on the alkaline shift in the pH(i) dependence of Na+/H+ exchange and that deletion of region A also abolished such an alkaline shift induced by a short thrombin stimulation, The results st rongly suggest that the early thrombin response and the ionomycin resp onse share the same activation mechanism. Based on these data and the results shown in the accompanying paper (Bertrand, B., Wakabayashi, S. , Ikeda, T, Pouyssegur, J., and Shigekawa, M. (1994) J. Biol. Chem. 26 9, 13703-13709), we propose that CaM is one of the major ''signal tran sducers'' that mediate distinct extracellular signals to the ''pH(i) s ensor'' of NHE1.