![]() Structure determination of human cardiac NCX1 Here we present the structures of human cardiac NCX1 in both the inactivated and Ca 2+-activated states, revealing the molecular basis underlying the complex regulation of NCX1. NCX1 activity can also be modulated by PIP2 36, 42, 43, 44 as well as posttranslational modification such as palmitoylation 45, 46, 47, 48.ĭespite the extensive investigations, little is known about the architecture of the eukaryotic NCX1 and the coupling between its TM and intracellular regulatory domains. An amphipathic region, known as eXchanger Inhibitory Peptide (XIP) at the N-terminus of the intracellular domain, plays a central role in the inactivation process 40, 41. The Na +-dependent inactivation occurs when three Na + ions are bound to the inward-facing exchanger 37, 38, 39. In cardiac NCX1, cytosolic Ca 2+ binds to two calcium-binding domains (CBDs) within the intracellular domain to increase transport activity 33, 34, 35, while cytosolic Na + inhibits transport activity by driving the exchanger into an inactivated state known as Na +-dependent inactivation 16, 31, 36, 37. The intracellular domain is involved in the allosteric regulation of the exchanger by cytosolic Ca 2+ and Na + 29, 31. The TM domain is responsible for the ion exchange function in NCX 29, 31 and has been modeled by the crystal structures of the archaebacterial exchanger NCX_Mj 30, 32. The eukaryotic NCX consists of a TM domain with 10 TM helices and a large intracellular regulatory domain which, in primary sequence, separates the TM domain into two homologous halves (TMs 1–5 and TMs 6–10) 2, 27, 28, 29, 30. The cardiac exchanger NCX1.1 has been the most extensively studied variant whose function is central to the cardiac contractile activity 23, 24, 25, 26. Three NCX isoforms (NCX1–3) are found in mammals, and each isoform carries various splice variants that are expressed in specific tissues and exhibit distinct regulatory phenotypes 2, 17, 18, 19, 20, 21, 22. NCX normally functions to extrude Ca 2+, but it can be reversed depending on the chemical gradient of Na + and Ca 2+ and the membrane potential 1, 10, 11, 12, 13, 14, 15, 16. NCX-catalyzed ion exchange reaction is electrogenic with a stoichiometry of 3 Na + for 1 Ca 2+. Dysfunctions of NCXs are associated with many human pathologies, including cardiac hypertrophy, arrhythmia, and postischemic brain damage 1, 7, 8, 9. They are ubiquitously expressed and play a central role in maintaining cellular calcium homeostasis for cell signaling 5, 6. Sodium-calcium exchangers (NCX) are membrane antiporters that control the extrusion or entry of Ca 2+ across the cell membrane in exchange for Na + 1, 2, 3, 4. Thus, the current NCX1 structures provide an essential framework for the mechanistic understanding of the ion transport and cellular regulation of NCX family proteins. Ca 2+ binding to the cytosolic second Ca 2+-binding domain (CBD2) disrupts this inactivation assembly which releases its constraint on the TM domain, yielding an active exchanger. In the inward-facing state with low cytosolic, a TM-associated four-stranded β-hub mediates a tight packing between the TM and cytosolic domains, resulting in the formation of a stable inactivation assembly that blocks the TM movement required for ion exchange function. ![]() We demonstrate that the interactions between the ion-transporting transmembrane (TM) domain and the cytosolic regulatory domain define the activity of NCX. Here we present the cryo-EM structures of human cardiac NCX1 in both inactivated and activated states, elucidating key structural elements important for NCX ion exchange function and its modulation by cytosolic Ca 2+ and Na +. Although extensively studied, little is known about the global structural arrangement of eukaryotic NCXs and the structural mechanisms underlying their regulation by various cellular cues including cytosolic Na + and Ca 2+. They are ubiquitously expressed and play an essential role in maintaining cytosolic Ca 2+ homeostasis. ![]() Is accompanied by a release of energy in the form of heat.Na +/Ca 2+ exchangers (NCX) transport Ca 2+ in or out of cells in exchange for Na +.
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