Direct Loading of the purified endogenous inhibitor into the cytoplasm of patched cardiomyocytes blocks the ion currents and calcium transport through the NCX1 protein.
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.
Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel.
Liron
L
Boyman
Reuben
R
Hiller
W Jonathan
WJ
Lederer
Daniel
D
Khananshvili
D000818:Animals; D001685:Biological Factors / Q000302:isolation & purification* / Q000493:pharmacokinetics / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000493:pharmacokinetics; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D017136:Ion Transport / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000502:physiology*
D001685:Biological Factors; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D002118:Calcium