Local Control of Cardiac Sodium-Calcium Exchanger by PMCA in Submembrane Microdomain in Mouse Heart Cells

Abstract

OBJECT: This study aimed at unveiling functional interaction between the Na/Ca exchanger (NCX) and the plasma membrane Ca2+-ATPase (PMCA) on the sarcolemmal membrane of heart cells. METHOD: The Na/Ca exchange current (INCX) was recorded from whole-cell clamped mouse ventricular myocytes under physiological conditions at 37°C. Functions of the ryanodine receptor and SERCA Ca2+-pump on the sarcoplasmic reticulum were abolished by using ryanodine and thapsigargin. The INCX was isolated as Ni2+-sensitive current component, under the conditions that eliminate other major membrane current systems. RESULTS: With the [Ca2+]i strongly buffered with 10 mM-BAPTA, the INCX was recorded as a time-independent current. However, with the [Ca2+]i only weakly buffered with 0.1 mM-BAPTA, the INCX showed a small current amplitude and a slow activation time-course. This was not observed when the [Ca2+]i was strongly buffered (with 10 mM-BAPTA). Inhibition of PMCA by intracellular administration of orthovanadate (VO43-) dramatically increased the amplitude of INCX and accelerated its activation kinetics. At the same time, orthovanadate shifted the [Ca2+]i-dependence of the INCX amplitude (EC50 = 0.97 µM) to lower levels (EC50 = 0.40 µM). Moreover, similar effects were observed by intracellular application of a selective PMCA inhibitor 5(6)-carboxyeosin. CONCLUSION: The PMCA regulates the operation of the NCX by altering local [Ca2+]i level around the NCX molecule. Because PMCA is driven by ATP, this functional coupling might serve as a mechanism that the intracellular metabolic status to the [Ca2+]i regulation and Ca2+ signaling in heart cells.