- MeSH
- Biological Transport physiology MeSH
- Research Support as Topic MeSH
- Homeostasis MeSH
- Rabbits MeSH
- Rats MeSH
- Myocardium MeSH
- Cell Membrane Permeability MeSH
- Sodium-Calcium Exchanger physiology MeSH
- Mammals MeSH
- Animals MeSH
- Check Tag
- Rabbits MeSH
- Rats MeSH
- Animals MeSH
- Publication type
- Review MeSH
- Comparative Study MeSH
The aim of this study was to examine the relationship between sarcolemmal Na(+)-Ca2+ exchangers and sarcoplasmic reticulum (SR) Ca(2+) -ATPase (SERCA2) expression and the developmental differences in cardiac Ca2+ handling. Postnatal steady-state mRNA and protein levels were analysed in rat ventricular myocardium by Northern and immunoblot analysis, respectively. This was compared to Na+ gradient-induced and SR oxalate-supported Ca2 transport in isolated membranes. Na(+)-Ca2+ exchanger mRNA declined by 75% between day 1 and 30, whereas SR Ca2+ ATPase mRNA levels increased by 97% during this period. The Na(+)-Ca2+ exchanger mRNA/Ca(2+)-ATPase mRNA ratio was found to be inversely related to post-natal age. The changes in mRNA levels were associated with corresponding developmental differences in the Ca2+ transport activities of the respective membrane proteins. In crude membranes, the Na(+)-dependent Ca2+ transport activity (at 75 microM Ca2+) declined gradually (P < 0.01; mean +/- S.E.) from 17.7 +/- 2.4 nmoles Ca2+/g wet tissue/2s at day 1-3 (n = 5) to a value of 4.2 +/- 1.1 at day 40 (n =4). Conversely, SR Ca2+ uptake increased (P < 0.01) 2.6-fold during this period. The inversely related changes in the post-natal expression and function of the Na(+)-Ca2+ exchanger and SR Ca(2+)-ATPase suggest a coordinated control at the pretranslational level of the cellular Ca2+ transport processes mediated by the two membrane proteins.
- MeSH
- Calcium-Transporting ATPases biosynthesis MeSH
- Gene Expression * MeSH
- Rats MeSH
- RNA, Messenger biosynthesis metabolism MeSH
- Myocardium * metabolism MeSH
- Blotting, Northern MeSH
- Oxalates pharmacology MeSH
- Rats, Wistar MeSH
- Sarcolemma * metabolism MeSH
- Sarcoplasmic Reticulum * metabolism MeSH
- Heart physiology growth & development MeSH
- Heart Rate MeSH
- Aging * metabolism MeSH
- In Vitro Techniques MeSH
- Carrier Proteins * biosynthesis MeSH
- Calcium * metabolism MeSH
- Blotting, Western MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
Na(+)/Ca(2+) exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj (Methanococcus jannaschii NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na(+) or Ca(2+) specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity (Km ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.
- MeSH
- Apoproteins chemistry genetics metabolism MeSH
- Archaeal Proteins chemistry genetics metabolism MeSH
- Cell Membrane chemistry MeSH
- Cysteine chemistry MeSH
- Protein Interaction Domains and Motifs MeSH
- Mutagenesis, Insertional MeSH
- Kinetics MeSH
- Protein Conformation MeSH
- Ligands MeSH
- Methanocaldococcus metabolism MeSH
- Models, Molecular * MeSH
- Mutation MeSH
- Peptide Fragments chemistry genetics metabolism MeSH
- Sodium-Calcium Exchanger chemistry genetics metabolism MeSH
- Recombinant Proteins chemistry metabolism MeSH
- Sodium metabolism MeSH
- Protein Stability MeSH
- Amino Acid Substitution MeSH
- Calcium metabolism MeSH
- Binding Sites MeSH
- Deuterium Exchange Measurement MeSH
- Computational Biology MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
