Dataset Information

Structural dynamics of Na⁺ and Ca²⁺ interactions with full-size mammalian NCX.

ABSTRACT: Cytosolic Ca²⁺ and Na⁺ allosterically regulate Na⁺/Ca²⁺ exchanger (NCX) proteins to vary the NCX-mediated Ca²⁺ entry/exit rates in diverse cell types. To resolve the structure-based dynamic mechanisms underlying the ion-dependent allosteric regulation in mammalian NCXs, we analyze the apo, Ca²⁺, and Na⁺-bound species of the brain NCX1.4 variant using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Ca²⁺ binding to the cytosolic regulatory domains (CBD1 and CBD2) rigidifies the intracellular regulatory loop (5L6) and promotes its interaction with the membrane domains. Either Na⁺ or Ca²⁺ stabilizes the intracellular portions of transmembrane helices TM3, TM4, TM9, TM10, and their connecting loops (3L4 and 9L10), thereby exposing previously unappreciated regulatory sites. Ca²⁺ or Na⁺ also rigidifies the palmitoylation domain (TMH2), and neighboring TM1/TM6 bundle, thereby uncovering a structural entity for modulating the ion transport rates. The present analysis provides new structure-dynamic clues underlying the regulatory diversity among tissue-specific NCX variants.

SUBMITTER: Giladi M

PROVIDER: S-EPMC11021524 | biostudies-literature | 2024 Apr

REPOSITORIES: biostudies-literature

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Structural dynamics of Na+ and Ca2+ interactions with full-size mammalian NCX.

Giladi Moshe M Fojtík Lukáš L Strauss Tali T Da'adoosh Benny B Hiller Reuben R Man Petr P Khananshvili Daniel D

Communications biology 20240416 1

Cytosolic Ca2+ and Na+ allosterically regulate Na+/Ca2+ exchanger (NCX) proteins to vary the NCX-mediated Ca2+ entry/exit rates in diverse cell types. To resolve the structure-based dynamic mechanisms underlying the ion-dependent allosteric regulation in mammalian NCXs, we analyze the apo, Ca2+, and Na+-bound species of the brain NCX1.4 variant using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular d ...[more]

PMID: 38627576

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Project description:BackgroundWhile calcium is known to play a crucial role in mammalian sperm physiology, how it flows in and out of the male gamete is not completely understood. Herein, we investigated the involvement of Na+/Ca2+ exchangers (NCX) in mammalian sperm capacitation. Using the pig as an animal model, we first confirmed the presence of NCX1 and NCX2 isoforms in the sperm midpiece. Next, we partially or totally blocked Ca2+ outflux (forward transport) via NCX1/NCX2 with different concentrations of SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; 0, 0.5, 5 and 50 µM) and Ca2+ influx (reverse transport) with SN6 (ethyl 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-1,3-thiazolidine-4-carboxylate; 0, 0.3, 3 or 30 µM). Sperm were incubated under capacitating conditions for 180 min; after 120 min, progesterone was added to induce the acrosome reaction. At 0, 60, 120, 130, and 180 min, sperm motility, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), tyrosine phosphorylation of sperm proteins, and intracellular levels of Ca2+, reactive oxygen species (ROS) and superoxides were evaluated.ResultsPartial and complete blockage of Ca2+ outflux and influx via NCX induced a significant reduction of sperm motility after progesterone addition. Early alterations on sperm kinematics were also observed, the effects being more obvious in totally blocked than in partially blocked samples. Decreased sperm motility and kinematics were related to both defective tyrosine phosphorylation and mitochondrial activity, the latter being associated to diminished MMP and ROS levels. As NCX blockage did not affect the lipid disorder of plasma membrane, the impaired acrosome integrity could result from reduced tyrosine phosphorylation.ConclusionsInhibition of outflux and influx of Ca2+ triggered similar effects, thus indicating that both forward and reverse Ca2+ transport through NCX exchangers are essential for sperm capacitation.

Dataset Information

Structural dynamics of Na⁺ and Ca²⁺ interactions with full-size mammalian NCX.

Publications

Structural dynamics of Na<sup>+</sup> and Ca<sup>2+</sup> interactions with full-size mammalian NCX.

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