Cross-talk between native plasmalemmal Na+/Ca2+ exchanger and inositol 1,4,5-trisphosphate-sensitive ca2+ internal store in Xenopus oocytes.
Because the presence of a native plasmalemmal Na+/Ca2+ 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 (InsP3R) that controls intracellular Ca2+ 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 InsP3Rs 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 Ca2+ mediated by InsP3, voltage-clamped oocytes display a large and transient inward Ca2+ -activated chloride current, IClCa, as a result of the Ca2+ 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 "Ca2+ extrusion" via NCX; and (v) blocking NCX enhanced the IClCa elicited by InsP3 but not by Ca2+ photolysis in oocytes injected with the respective caged compounds. Moreover, overexpression of cardiac NCX1, confirmed by confocal microscopy, has functional consequences for the "Ca2+ influx" but not for the serum-elicited "Ca2+ efflux" mode of basal exchange activity and does not alter the number of endogenous NCX/InsP3Rs colocalization sites. Our results suggest that native NCX, because of its strategic position, may regulate InsP3-mediated Ca2+ signaling during the early phases of oocyte maturation and/or fertilization, and furthermore foreign cardiac protein is excluded from the Ca2+ microdomains surrounding the native NCX/InsP3Rs complex in the oocyte.
Solís-Garrido
Luisa M
LM
Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, 28029 Madrid, Spain.
Pintado
Antonio J
AJ
Andrés-Mateos
Eva
E
Figueroa
María
M
Matute
Carlos
C
Montiel
Carmen
C
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D018075:RNA, Complementary / Q000235:genetics; D020239:Receptor Cross-Talk; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D014982:Xenopus laevis
C101670:2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate; D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018075:RNA, Complementary; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D015544:Inositol 1,4,5-Trisphosphate; D013890:Thiourea; D002118:Calcium