Direct Loading of the Purified Endogenous Inhibitor into the Cytoplasm of Patched Cardiomyocytes Blocks the Ion Currents and Calcium Transport through the NCX1 Protein

Abstract

The Na(+)-Ca(2+) exchanger in mammalian heart muscle (NCX1) is the central transporter protein that regulates extrusion of Ca(2+) from the heart cell. However, the functional biochemistry and physiology of NCX1 have been severely hampered by the absence of any specific high-affinity inhibitor. Here we describe advanced procedures for purifying a candidate inhibitor, previously called endogenous inhibitor factor (NCX(IF)), and demonstrate its direct actions on NCX1 activities in the single-cell system. A combination of advanced HILIC (hydrophilic interaction liquid chromatography) procedures with analytical tests suggests that the properties of NCX(IF) resemble those of a small (disaccharide size) polar molecule lacking any aromatic rings, conjugated bonds, or a primary amino group. The effects of NCX(IF) on the NCX1-mediated ion currents (I(NCX)) and cytosolic Ca(2+) extrusion were detected by a combination of patch-clamp and confocal microscopy under conditions in which the purified NCX(IF) was directly loaded into the cytoplasm of patched cardiomyocytes. It was demonstrated that cytosolic NCX(IF) blocks the Ca(2+)-activated NCX1 inward current and the accompanying extrusion of Ca(2+) from the cell with high efficacy. A constant fraction of NCX1 inhibition was observed under conditions in which the cytosolic [Ca(2+)](i) was varied at fixed doses of NCX(IF), suggesting that the degree of inhibition is controlled by NCX(IF) dose and not by cytosolic Ca(2+) concentration. NCX(IF) blocks equally well both the Ca(2+) extrusion and Ca(2+) entry modes of NCX1, consistent with thermodynamic principles expected for the functioning of a bidirectional "carrier-type" transport system. We concluded that NCX(IF) interacts with a putative regulatory domain from the cytosolic side and, thus, may play an important regulatory role in controlling Ca(2+) signaling in the heart. This may represent a new potential tool for developing novel treatments for cardiac Ca(2+) signaling dysfunction.

Calcium Movement in Cardiac Mitochondria

“…A detailed description of the materials and methods used here can be found in the expanded Supporting Material. Briefly, rat ventricular myocytes were isolated using standard enzymatic approaches (27). Electrophysiological experiments were performed using standard protocols with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA).…”

Essential Role of the CBD1-CBD2 Linker in Slow Dissociation of Ca2+ from the Regulatory Two-domain Tandem of NCX1

“…free , and the data were fitted to an Adair or Hill equation using the GraFit software (Erithacus Software Ltd.) as described before (21). All other miscellaneous procedures were described elsewhere (38,39).…”

“…Experimentally obtained traces were fitted to mono-exponential or multi-exponential curves by using the 2 criteria to identify the best fit for a specific kinetic model of Ca 2ϩ dissociation. All other details for data processing and analysis are described elsewhere (35)(36)(37)(38)40).…”

Proton-sensing Ca2+ Binding Domains Regulate the Cardiac Na+/Ca2+ Exchanger

“…Cell Isolation, Electrophysiology, and Confocal ImagingCardiomyocytes were isolated from euthanized adult SpragueDawley rats (18). Myocytes were attached to laminin-coated coverslips placed in a custom designed chamber and were used within 4 -5 h from the time of isolation.…”