Cross-talk between native plasmalemmal Na+/Ca2+ exchanger and inositol 1,4,5-trisphosphate-sensitive Ca2+ internal store in Xenopus oocytes

Abstract

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 3R) that controls intracellular Ca2+ in a restricted subplasmalemmal space. The follow-ing 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, ICl(Ca), 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 ICl(Ca) elicited by InsP 3 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 "Ca 2+ 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 InsP 3-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.