Actin-dependent regulation of the cardiac Na(+)/Ca(2+) exchanger.
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.
Condrescu
Madalina
M
Department. of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 South Orange Ave., PO Box 1709, Newark, NJ 07101-1709, USA.
Reeves
John P
JP
D000199:Actins / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D006224:Cricetinae; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D021381:Protein Transport; D019831:Sodium-Calcium Exchanger / Q000235:genetics / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000494:pharmacology
D000199:Actins; D019831:Sodium-Calcium Exchanger; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D015638:Cytochalasin D; D002118:Calcium