Time-resolved monitoring of electrogenic Na+-Ca2+ exchange in the isolated cardiac sarcolemma vesicles by using a rapid-response fluorescent probe - PubMed (original) (raw)
. 1999 Feb 2;38(5):1435-45.
doi: 10.1021/bi981429u.
Affiliations
- PMID: 9931008
- DOI: 10.1021/bi981429u
Time-resolved monitoring of electrogenic Na+-Ca2+ exchange in the isolated cardiac sarcolemma vesicles by using a rapid-response fluorescent probe
D Baazov et al. Biochemistry. 1999.
Abstract
As a major Ca exit system in myocytes, the electrogenic Na+-Ca2+ exchange is exposed to rapid changes of regulatory factors (e.g., cytosolic Ca) during the excitation-contraction coupling. The dynamic aspects of the exchanger response to regulatory factors have not been resolved in the past due to technical limitations. Here, we describe stopped-flow protocols for monitoring the electrogenic activity of Na+-Ca2+ exchange in cardiac sarcolemma vesicles by using a rapid-response voltage-sensitive dye Merocyanine-540 (M540). The M540 signal of Nao-dependent Ca efflux is generated by mixing the Ca-loaded vesicles with Na buffer, yielding 160 mM extravesicular Na and 6 microM Cafree. This signal is inhibited by a cyclic peptide blocker (FRCRCFa), by a Ca ionophore (ionomycin), or by an electrogenic uncoupler (valinomycin or FCCP). The M540 signal of Nao-dependent Ca efflux shows a rapid pre-steady-state burst (210 s-1), followed by slow steady-state phase (</=5 s-1). Extravesicular (cytosolic) Ni inhibits both phases with an IC50 of 0.80 +/- 0.24 mM. At an extravesicular pH of 6.0, the Nao-dependent Ca efflux is able to generate the M540 signal, thereby supporting the idea that the stoichiometry of Na+-Ca2+ exchange is not altered at low pH [Khanashvili, D., et al. (1995) Biochemistry 34, 10290-10297]. The M540 signal of Nao-dependent Ca efflux is lost when the extravesicular Cafree concentration drops to 0.2 microM. This effect cannot be explained by a lack of Ca access to extravesicular (cytosolic) transport sites, because the reaction of Nao-dependent Ca efflux utilizes intravesicular Ca as a substrate. These data suggest that in sarcolemma vesicles a regulatory cytosolic Ca site controls the exchanger activity. The properties of this putative regulatory site do not resemble the properties of the "slow" Ca regulatory mode, observed in electrophysiological studies. Under saturating ionic conditions, the Nao-dependent Ca efflux generates the initial rates of 21 mV/ms in the vesicles with a diameter of 3000-5000 A. If a site density of 300-400 exchangers/micrometer2 and a vesicular surface of 0.5 micrometer2 are assumed, each vesicle may contain 150-200 exchanger molecules with a maximal turnover rate of 4000-5000 s-1. This upper limit for turnover (no matter what the site density is) may put considerable restrictions on the exchanger capacity to mediate Ca entry in the cell under physiologically related conditions.
Similar articles
- Rapid interaction of FRCRCFa with the cytosolic side of the cardiac sarcolemma Na(+)-Ca2+ exchanger blocks the ion transport without preventing the binding of either sodium or calcium.
Khananshvili D, Baazov D, Weil-Maslansky E, Shaulov G, Mester B. Khananshvili D, et al. Biochemistry. 1996 Dec 10;35(49):15933-40. doi: 10.1021/bi961099i. Biochemistry. 1996. PMID: 8961960 - Positively charged cyclic hexapeptides, novel blockers for the cardiac sarcolemma Na(+)-Ca2+ exchanger.
Khananshvili D, Shaulov G, Weil-Maslansky E, Baazov D. Khananshvili D, et al. J Biol Chem. 1995 Jul 7;270(27):16182-8. doi: 10.1074/jbc.270.27.16182. J Biol Chem. 1995. PMID: 7608184 - The cardiac Na(+)-Ca2+ exchanger: relative rates of calcium and sodium movements and their modulation by protonation-deprotonation of the carrier.
Khananshvili D, Weil-Maslansky E. Khananshvili D, et al. Biochemistry. 1994 Jan 11;33(1):312-9. doi: 10.1021/bi00167a041. Biochemistry. 1994. PMID: 8286352 - Electrogenic Na+/Ca2+-exchange of nerve and muscle cells.
Török TL. Török TL. Prog Neurobiol. 2007 Aug;82(6):287-347. doi: 10.1016/j.pneurobio.2007.06.003. Epub 2007 Jun 21. Prog Neurobiol. 2007. PMID: 17673353 Review. - Low-sodium contractures indicating sarcolemmal Na/Ca-exchange in the frog heart.
Volkmann R. Volkmann R. Comp Biochem Physiol A Comp Physiol. 1988;91(2):225-34. doi: 10.1016/0300-9629(88)90409-4. Comp Biochem Physiol A Comp Physiol. 1988. PMID: 2904338 Review.
Cited by
- Structural dynamics of Na+ and Ca2+ interactions with full-size mammalian NCX.
Giladi M, Fojtík L, Strauss T, Da'adoosh B, Hiller R, Man P, Khananshvili D. Giladi M, et al. Commun Biol. 2024 Apr 16;7(1):463. doi: 10.1038/s42003-024-06159-9. Commun Biol. 2024. PMID: 38627576 Free PMC article. - Conformational free-energy landscapes of a Na+/Ca2+ exchanger explain its alternating-access mechanism and functional specificity.
Marinelli F, Faraldo-Gómez JD. Marinelli F, et al. Proc Natl Acad Sci U S A. 2024 Apr 16;121(16):e2318009121. doi: 10.1073/pnas.2318009121. Epub 2024 Apr 8. Proc Natl Acad Sci U S A. 2024. PMID: 38588414 Free PMC article. - Na+/Ca2+ exchange in enamel cells is dominated by the K+-dependent NCKX exchanger.
Souza Bomfim GH, Mitaishvili E, Schnetkamp PPM, Lacruz RS. Souza Bomfim GH, et al. J Gen Physiol. 2024 Jan 1;156(1):e202313372. doi: 10.1085/jgp.202313372. Epub 2023 Nov 10. J Gen Physiol. 2024. PMID: 37947795 Free PMC article. - Structure-Based Function and Regulation of NCX Variants: Updates and Challenges.
Khananshvili D. Khananshvili D. Int J Mol Sci. 2022 Dec 21;24(1):61. doi: 10.3390/ijms24010061. Int J Mol Sci. 2022. PMID: 36613523 Free PMC article. Review. - Proton-modulated interactions of ions with transport sites of prokaryotic and eukaryotic NCX prototypes.
Refaeli B, Liu S, Hiller R, Giladi M, Baiz CR, Khananshvili D. Refaeli B, et al. Cell Calcium. 2021 Nov;99:102476. doi: 10.1016/j.ceca.2021.102476. Epub 2021 Sep 20. Cell Calcium. 2021. PMID: 34564055 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Research Materials
Miscellaneous