Regulation of Na+/H+ Exchanger Isoform 1 (NHE1) by Calmodulin-binding Sites: Role of Angiotensin II
We examined the effect of Angiotensin II (Ang II) on the interaction between the Ca<sup>2+</sup>/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<sup>1K3R/4E</sup> and SB<sup>1K3R/4E</sup>. Wild type and mutants were transfected into PS120 cells and their activity was examined by H<sup>+</sup> flux (J<sub>H+</sub>). The basal J<sub>H+</sub> 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<sup>-12</sup> and 10<sup>-9</sup> M) increased the J<sub>H+</sub> in wild type and SB. Ang II (10<sup>-6</sup> M) increased this parameter only in SA. Ang II (10<sup>-9</sup> M) maintained the expression of calmodulin in wild type or mutants, and Ang II (10<sup>-6</sup> M) decreased it in wild type or SA, but not in SB. Dimethyl-Bapta-AM (10<sup>-7</sup> M), a calcium chelator, suppressed the effect of Ang II (10<sup>-9</sup> M) in wild type. With Ang II (10<sup>-6</sup> M), Bapta failed to affect wild type or SA, but it increased the J<sub>H+</sub> in SB. W13 or calmidazolium chloride (10<sup>-5</sup> M), two distinct calmodulin inhibitors, decreased the effect of Ang II (10<sup>-9</sup> M) in wild type or SB. With Ang II (10<sup>-6</sup> M), W13 or calmidazolium chloride decreased the J<sub>H+</sub> in wild type or SA and increased it in SB. Thus, with Ang II (10<sup>-12</sup> and 10<sup>-9</sup> M), site A seems to be responsible for the stimulation of hNHE1 and with Ang II (10<sup>-6</sup> M), site B is important to maintain its basal activity.