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.

Action of ANP on the nongenomic dose-dependent biphasic effect of aldosterone on NHE1 in proximal S3 segment

“…A low affinity site, however, binds to Ca 2+ and calmodulin only at high concentrations and, under these conditions, inhibits the exchanger activity. More recently, we modified amino acids in these two binding sites of NHE1 by site-directed mutagenesis and obtain data that reinforce this idea [41]. This behavior is compatible with our present findings, indicating stimulation of the NHE1 exchanger by increases of [Ca 2+ ] i in the lower range (at 10 −12 M ALDO) and inhibition of this exchanger at high [Ca 2+ ] i levels (at 10 −6 M ALDO).…”

Dose-dependent effects of angiotensin-(1–7) on the NHE3 exchanger and [Ca2+]iin in vivo proximal tubules

“…As BAPTA alone decreases the basal [Ca 2ϩ ] i but does not change the basal JHCO 3 Ϫ , it seems difficult to compare the responses with ANG-(1-7) plus BAPTA. However, BAPTA decreases the inhibitory effect of ANG-(1-7) (10 Ϫ9 M) on JHCO 3 Ϫ and impairs the stimulatory effect of ANG-(1-7) (10 Ϫ6 M) on JHCO 3 Ϫ because it impairs the stimulatory effect of ANG-(1-7) (10 Ϫ9 or 10 Ϫ6 M) on the [Ca 2ϩ ] i (7,43). On the other hand, thapsigargin alone decreases the JHCO 3 Ϫ , maintains the inhibitory effect of ANG-(1-7) (10 Ϫ9 M) on JHCO 3 Ϫ , and changes the stimulatory effect of ANG-(1-7) (10 Ϫ6 M) on JHCO 3 Ϫ to an inhibitory effect because, in these three conditions, thapsigargin increases the [Ca 2ϩ ] i to about 3,5 times the control value, which by itself inhibits the NHE exchanger.…”

“…In addition, it has been shown that the NHE exchanger has calmodulin binding sites at the cytoplasmatic regulatory domain, which modulate its activity (7,43). Confirming this finding, studies from our laboratory have shown that the dose-dependent biphasic effects of ANG II (32,34), aldosterone (26), and arginine vasopressin (33) on the Na ϩ /H ϩ exchanger are associated with either a small or large increase in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ).…”

High Glucose Concentration Stimulates NHE-1 Activity in Distal Nephron Cells: the Role of the Mek/Erk1/2/p90RSKand p38MAPK Signaling Pathways

“…As described in our previous studies [27,28,29], intracellular pH (pH i ) was monitored using the BCECF-AM fluorescent probe (Molecular Probes, Eugene, OR, USA). Cells grown to confluence on glass coverslips were fluorescently labeled using 5 µM BCECF-AM in control solution [28] for 3 minutes.…”