The cardiac Na⁺-Ca²⁺ exchanger has two cytoplasmic ion permeation pathways

The Na⁺-Ca²⁺ exchanger (NCX) is a ubiquitously expressed plasma membrane protein. It plays a fundamental role in Ca ²⁺ homeostasis by moving Ca²⁺ out of the cell using the electrochemical gradient of Na⁺ as the driving force. Recent structural studies of a homologous archaebacterial exchanger, NCX-Mj, revealed its outward configuration with two potential ion permeation pathways exposed to the extracellular environment. Based on the symmetry of NCX-Mj structure, an atomic model of an inward-facing conformation was generated showing similar pathways but directed to the cytoplasm. The presence of these water-filled cavities has yet to be confirmed experimentally, and it is unknown if the mammalian exchanger adopts the same structure. In this study, we mutated multiple residues within transmembrane segments 2 and 7 of NCX1.1 (cardiac isoform) to cysteines, allowing us to investigate their sensitivity to membrane-impermeable sulfhydryl reagents as exchanger current block. By trapping NCX1.1 in the inward-facing configuration, we have mapped two differently sized cytoplasmic aqueous cavities, the access of which is modified during exchange. This data reveals movements of the protein associated with ion transport. Electrophysiological characterization shows that the conserved residues within transmembrane segments 2 and 7, coordinating Na⁺ and Ca²⁺ ions in NCX-Mj, play a fundamental role in NCX1.1. Our results suggest a similar architecture between the mammalian and archaebacterial exchangers.