Abstract
In the present study, the bovine cardiac Na$^+$/Ca$^2+$ exchanger
(NCX1.1) was expressed in Chinese hamster ovary cells. The surface
distribution of the exchanger protein, externally tagged with the
hemagglutinin (HA) epitope, was associated with underlying actin
filaments in regions of cell-to-cell contact and also along stress
fibers. After we treated cells with cytochalasin D, NCX1.1 protein
colocalized with patches of fragmented filamentous actin (F-actin).
In contrast, an HA-tagged deletion mutant of NCX1.1 that was missing
much of the exchanger's central hydrophilic domain Delta(241-680)
did not associate with F-actin. In cells expressing the wild-type
exchanger, cytochalasin D inhibited allosteric Ca$^2+$ activation
of NCX activity as shown by prolongation of the lag phase of low
Ca$^2+$ uptake after initiation of the reverse (i.e., Ca$^2+$
influx) mode of NCX activity. Other agents that perturbed F-actin
structure (methyl-beta-cyclodextrin, latrunculin B, and jasplakinolide)
also increased the duration of the lag phase. In contrast, when reverse-mode
activity was initiated after allosteric Ca$^2+$ activation, both
cytochalasin D and methyl-beta-cyclodextrin (Me-beta-CD) stimulated
NCX activity by approximately 70\%. The activity of the Delta(241-680)
mutant, which does not require allosteric Ca$^2+$ activation,
was also stimulated by cytochalasin D and Me-beta-CD. The increased
activity after these treatments appeared to reflect an increased
amount of exchanger protein at the cell surface. We conclude that
wild-type NCX1.1 associates with the F-actin cytoskeleton, probably
through interactions involving the exchanger's central hydrophilic
domain, and that this association interferes with allosteric Ca$^2+$
activation.
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