Regulation of Na+/H+ Exchanger Isoform 1 (NHE1) by Calmodulin-binding Sites: Role of Angiotensin II

Abstract

We examined the effect of Angiotensin II (Ang II) on the interaction between the Ca²⁺/CaM complex and hNHE1. Considering that calmodulin binds to NHE1 at two sites (A and B), amino acids at both sites were modified and two mutants were constructed: SA^1K3R/4E and SB^1K3R/4E. Wild type and mutants were transfected into PS120 cells and their activity was examined by H⁺ flux (J_H+). The basal J_H+ of wild type was 4.71 ± 0.57 (mM/min), and it was similar in both mutants. However, the mutations partially impaired the binding of CaM to hNHE1. Ang II (10^-12 and 10^-9 M) increased the J_H+ in wild type and SB. Ang II (10^-6 M) increased this parameter only in SA. Ang II (10^-9 M) maintained the expression of calmodulin in wild type or mutants, and Ang II (10^-6 M) decreased it in wild type or SA, but not in SB. Dimethyl-Bapta-AM (10^-7 M), a calcium chelator, suppressed the effect of Ang II (10^-9 M) in wild type. With Ang II (10^-6 M), Bapta failed to affect wild type or SA, but it increased the J_H+ in SB. W13 or calmidazolium chloride (10^-5 M), two distinct calmodulin inhibitors, decreased the effect of Ang II (10^-9 M) in wild type or SB. With Ang II (10^-6 M), W13 or calmidazolium chloride decreased the J_H+ in wild type or SA and increased it in SB. Thus, with Ang II (10^-12 and 10^-9 M), site A seems to be responsible for the stimulation of hNHE1 and with Ang II (10^-6 M), site B is important to maintain its basal activity.