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(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.
Department of Physiology and Biophysics, Instituto de Ciências Biomédicas, University of São Paulo, São Paulo, Brazil.
Débora Mai N
DM
Eguti
Karina
K
Thieme
George P
GP
Leung
Margarida
M
Mello-Aires
Maria
M
Oliveira-Souza
D019943:Amino Acid Substitution; D000804:Angiotensin II / Q000494:pharmacology / Q000502:physiology*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007093:Imidazoles / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D020033:Protein Isoforms / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D014162:Transfection
D002147:Calmodulin; D007093:Imidazoles; D020033:Protein Isoforms; D017923:Sodium-Hydrogen Exchangers; D013449:Sulfonamides; C069139:growth factor-activatable Na-H exchanger NHE-1; D000804:Angiotensin II; C031938:calmidazolium; C033772:N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide; D002118:Calcium