Expression of cardiac sarcolemmal Na+-Ca2+ exchange activity in Xenopus laevis oocytes

Because the presence of a native plasmalemmal Na ؉ / Ca 2؉ exchange (NCX) activity in Xenopus laevis oocytes remains controversial, its possible functional role in these cells is poorly understood. Here, in experiments on control oocytes and oocytes overexpressing a cloned NCX1 cardiac protein, confocal microscopy combined with electrophysiological techniques reveal that these cells express an endogenous NCX protein forming a functional microdomain with inositol 1,4,5-trisphosphate receptors (InsP 3 R) that controls intracellular Ca 2؉ in a restricted subplasmalemmal space. The following data obtained in control denuded oocytes are consistent with this view: (i) reverse transcription-PCR revealed that the oocyte expresses two transcripts for the NCX1 and NCX3 isoforms; (ii) immunofluorescence experiments showed that native NCX1 and InsP 3 Rs are largely codistributed in discrete areas of the plasma membrane in close apposition to the cortical endoplasmic reticulum shell; (iii) when stimulated by rabbit serum, which elevates intracellular Ca 2؉ mediated by InsP 3 , voltage-clamped oocytes display a large and transient inward Ca 2؉ -activated chloride current, I Cl(Ca) , as a result of the Ca 2؉ rise at the inner surface membrane; (iv) this current is significantly enhanced by KB-R7943 and by an extracellular sodium-depleted medium, two maneuvers that prevent "Ca 2؉ extrusion" via NCX; and (v) blocking NCX enhanced the I Cl(Ca) elicited by InsP 3 but not by Ca 2؉ photolysis in oocytes injected with the respective caged compounds. Moreover, overexpression of cardiac NCX1, confirmed by confocal microscopy, has functional consequences for the "Ca 2؉ influx" but not for the serum-elicited "Ca 2؉ efflux" mode of basal exchange activity and does not alter the number of endogenous NCX/InsP 3 Rs colocalization sites. Our results suggest that native NCX, because of its strategic position, may regulate InsP 3 -mediated Ca 2؉ signaling during the early phases of oocyte maturation and/or fertilization, and furthermore foreign cardiac protein is excluded from the Ca 2؉ microdomains surrounding the native NCX/InsP 3 Rs complex in the oocyte.

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“…8B). In fact, our data on the reverse-mode NCX activity presented here fit well with previous results obtained by other authors (10,12).…”

Section: Native Na ϩ /Ca 2ϩ Exchanger In Xenopus Oocytessupporting

confidence: 93%

Cross-talk between Native Plasmalemmal Na+/Ca2+ Exchanger and Inositol 1,4,5-Trisphosphate-sensitive Ca2+ Internal Store in Xenopus Oocytes

Solís-Garrido

Pintado

Andrés-Mateos

et al. 2004

Journal of Biological Chemistry

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“…Unincorporated nucleotides were removed by gel filtration on Chromaspin-100 columns (Clontech). Xenopus oocytes were injected with 46 nl of RNA, and exchanger activity was measured 1-5 days later as Na ϩ gradient-dependent 45 Ca 2ϩ uptake as described previously (20). Wild-type levels of activity varied between batches of oocytes from 5 to 50 pmol of Ca 2ϩ /oocyte taken up in 10 min.…”

Section: Methodsmentioning

confidence: 99%

Mutation of Amino Acid Residues in the Putative Transmembrane Segments of the Cardiac Sarcolemmal Na+-Ca2+ Exchanger

Nicoll

Hryshko

Matsuoka

et al. 1996

Journal of Biological Chemistry

136

151

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“…These cRNAs, in parallel with blank buffer, were microinjected into Xenopus oocytes (5 ng per oocyte). After 4-8 days of incubation, these oocytes were loaded with internal Na via nystatin (40), challenged with 45 Ca 2ϩ , washed, and assayed for 45 Ca 2ϩ uptake. Calcium uptake was induced by Calx cRNAs (Fig.…”

Section: Genetics: Schwarz and Benzermentioning

confidence: 99%

Calx, a Na-Ca exchanger gene ofDrosophila melanogaster

Schwarz

Benzer

1997

Proc. Natl. Acad. Sci. U.S.A.

196

157

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We have cloned Calx, a gene that encodes a Na-Ca exchanger of Drosophila melanogaster. Calx encodes two repeated motifs, Calx-␣ and Calx-␤, that overlap domains required for exchanger activity and regulation. Calx has multiple transcripts in adults, including at least one expressed in the retina. The Calx genomic locus comprises >35 kb between the Atp␣ and rudimentary-like genes in chromosomal region 93B. In Xenopus oocytes, microinjected Calx cRNA induces calcium uptake like that of its homolog, the 3Na ؉ -1Ca 2؉ exchanger of mammalian heart. Implications of Calx-␣ motifs for the mechanism of Na-Ca exchange are discussed.

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Expression of cardiac sarcolemmal Na+-Ca2+ exchange activity in Xenopus laevis oocytes

Cited by 39 publications

References 15 publications

Cross-talk between Native Plasmalemmal Na+/Ca2+ Exchanger and Inositol 1,4,5-Trisphosphate-sensitive Ca2+ Internal Store in Xenopus Oocytes

Cross-talk between Native Plasmalemmal Na+/Ca2+ Exchanger and Inositol 1,4,5-Trisphosphate-sensitive Ca2+ Internal Store in Xenopus Oocytes

Mutation of Amino Acid Residues in the Putative Transmembrane Segments of the Cardiac Sarcolemmal Na+-Ca2+ Exchanger

Calx, a Na-Ca exchanger gene ofDrosophila melanogaster

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