Proton-sensing Ca 2+ binding domains regulate the cardiac Na +/Ca 2+ exchanger

Abstract

The cardiac Na +/Ca 2+ exchanger (NCX) regulates cellular [Ca 2+] i and plays a central role in health and disease, but its molecular regulation is poorly understood. Here we report on how protons affect this electrogenic transporter by modulating two critically important NCX C 2 regulatory domains, Ca 2+binding domain-1 (CBD1) and CBD2. The NCX transport rate in intact cardiac ventricular myocytes was measured as a membrane current, I NCX, whereas [H +] i was varied using an ammonium chloride "rebound" method at constant extracellular pH 7.4. At pHi = 7.2 and [Ca 2+] i < 120 nM, I NCX was less than 4% that of its maximally Ca 2+-activated value. I NCX increases steeply at [Ca 2+] i between 130-150 nM with a Hill coefficient (n H) of 8.0 ± 0.7 and K 0.5 = 310 ± 5 nM. At pH i = 6.87, the threshold of Ca 2+- dependent activation of I NCX was shifted to much higher [Ca 2+] i (600-700 nM), and the relationship was similarly steep (n H = 8.0±0.8) with K 0.5 = 1042 ± 15 nM. The V max of Ca 2+-dependent activation of I NCX was not significantly altered by low pH i. The Ca 2+ affinities for CBD1 (0.39 ± 0.06 μM) and CBD2 (K d = 18.4 ± 6 μM) were exquisitely sensitive to [H +], decreasing 1.3-2.3-fold as pH i decreased from 7.2 to 6.9. This work reveals for the first time that NCX can be switched off by physiologically relevant intracellular acidification and that this depends on the competitive binding of protons to its C 2regulatory domains CBD1 and CBD2. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.