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 Ca 2+/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. © 2010 S. Karger AG Basel.