Inhibition of sodium-calcium exchange by KB-R7943: Dodecylamine and sphingosine in transfected Chinese hamster ovary cells.

Inhibitors of Na(+)/Ca(2+) exchange (NCX) such as KB-R7943 and SEA0400 are thought to act by promoting an inactive state of the exchanger induced by elevated cytosolic Na(+) concentrations (Na(+)-dependent inactivation). This conclusion is based mainly on experiments in excised patches from frog oocytes expressing NCX and has not been fully tested in intact cells. Here we characterize the inhibitory effects of KB-R7943 and other amphiphilic cations on NCX activity in transfected Chinese hamster ovary (CHO) cells expressing the cardiac isoform of the Na(+)/Ca(2+) exchanger (NCX1.

Calcineurin activation by slow calcium release from intracellular stores suppresses protein kinase C regulation of L-type calcium channels in L6 cells.

L-type Ca(2+) channel activity was assayed in L6 cells as the rate of nifedipine-sensitive Ba(2+) influx in a depolarizing medium. In the absence of extracellular Ca(2+), activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin (TMX) inhibited Ba(2+) influx by 38%. Thapsigargin (Tg), a selective inhibitor of the Ca(2+)-ATPase in the sarcoplasmic reticulum, evoked a rise in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in a Ca(2+)-free medium from 30 to approximately 80 nM.

Ionic regulation of the cardiac sodium-calcium exchanger.

The Na(+)-Ca(2+) exchanger (NCX) links transmembrane movements of Ca(2+) ions to the reciprocal movement of Na(+) ions. It normally functions primarily as a Ca(2+) efflux mechanism in excitable tissues such as the heart, but it can also mediate Ca(2+) influx under certain conditions. Na(+) and Ca(2+) ions exert complex regulatory effects on NCX activity.

Sodium-dependent inactivation of sodium/calcium exchange in transfected Chinese hamster ovary cells.

High concentrations of cytosolic Na(+) ions induce the time-dependent formation of an inactive state of the Na(+)/Ca(2+) exchanger (NCX), a process known as Na(+)-dependent inactivation. NCX activity was measured as Ca(2+) uptake in fura 2-loaded Chinese hamster ovary (CHO) cells expressing the wild-type (WT) NCX or mutants that are hypersensitive (F223E) or resistant (K229Q) to Na(+)-dependent inactivation. As expected, 1) Na(+)-dependent inactivation was promoted by high cytosolic Na(+) concentration, 2) the F223E mutant was more susceptible than the WT exchanger to inactivation, whereas the K229Q mutant was resistant, and 3) inactivation was enhanced by cytosolic acidification.

The sodium-calcium exchanger is a mechanosensitive transporter.

This report describes the influence of fluid flow and osmotically induced volume changes on Na(+)-Ca(2+) exchange (NCX) activity in transfected CHO cells. Exchange activity was measured as Na(+)-dependent Ca(2+) or Ba(2+) fluxes using the fluorescent probe fura-2. When exchange activity was initiated by superfusing Ba(2+)-containing solutions over the cells for a 20 s interval, a high rate of Ba(2+) uptake was observed while the solution was being applied but the rate of Ba(2+) uptake declined > 10-fold when the solution flow ceased.

New modes of exchanger regulation: physiological implications.

Exchange activity is regulated principally by cytosolic Na+, Ca2+, and PIP2. However, the properties of these modes of regulation that have emerged from excised patch studies appear to be poorly suited to regulating exchange activity on a beat-to-beat basis. Here we summarize recent findings from our lab indicating that (a) allosteric activation by Ca2+ exhibits hysteresis, (b) elevated concentrations of cytosolic Na+ induce a mode of activity that no longer requires regulatory Ca2+ activation, and (c) the requirement for PIP2 is reduced or eliminated after allosteric Ca2+ activation.

Regulation of the cardiac L-type calcium channel in L6 cells by arginine-vasopressin.

L-type Ca2+ channel activity was measured in L6 cells as nifedipine-sensitive barium (Ba2+; 5 mM) influx in a depolarizing salt solution containing 140 mM KCl. Addition of AVP (arginine-vasopressin) during Ba2+ uptake reduced the rate of Ba2+ influx by 60-100%; this was followed by a gradual restoration of the initial rate of Ba2+ uptake. Blockade of PKC (protein kinase C) by pretreatment with 10 muM bisindolylmaleimide did not affect the initial inhibition of Ba2+ influx, but completely abolished the recovery phase.

Sodium-calcium exchange does not require allosteric calcium activation at high cytosolic sodium concentrations.

The activity of the cardiac Na(+)-Ca(2+) exchanger (NCX1.1) is allosterically regulated by Ca(2+), which binds to two acidic regions in the cytosolically disposed central hydrophilic domain of the NCX protein. A mutation in one of the regulatory Ca(2+) binding regions (D447V) increases the half-activation constant (K(h)) for allosteric Ca(2+) activation from approximately 0.

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.

Allosteric activation of sodium-calcium exchange by picomolar concentrations of cadmium.

Chinese hamster ovary cells expressing the bovine cardiac Na+-Ca2+ exchanger (NCX1.1) accumulated Cd2+ after a lag period of several tens of seconds. The lag period reflects the progressive allosteric activation of exchange activity by Cd2+ as it accumulates within the cytosol.

Calcium-dependent regulation of calcium efflux by the cardiac sodium/calcium exchanger.

Allosteric regulation by cytosolic Ca2+ of Na(+)/Ca2+ exchange activity in the Ca2+ efflux mode has received little attention because it has been technically difficult to distinguish between the roles of Ca2+ as allosteric activator and transport substrate. In this study, we used transfected Chinese hamster ovary cells to compare the Ca2+ efflux activities in nontransfected cells and in cells expressing either the wild-type exchanger or a mutant, Delta(241-680), that operates constitutively; i.e.

Modulation of the voltage sensor of L-type Ca2+ channels by intracellular Ca2+.

Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, of L-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the concentrations that are expected to be near an open channel (>/=100 microM) completely and reversibly blocked calcium current through L-type channels.

Allosteric activation of sodium-calcium exchange activity by calcium: persistence at low calcium concentrations.

The activity of the cardiac Na+/Ca2+ exchanger is stimulated allosterically by Ca2+, but estimates of the half-maximal activating concentration have varied over a wide range. In Chinese hamster ovary cells expressing the cardiac Na+/Ca2+ exchanger, the time course of exchange-mediated Ca2+ influx showed a pronounced lag period followed by an acceleration of Ca2+ uptake. Lag periods were absent in cells expressing an exchanger mutant that was not dependent on regulatory Ca2+ activation.

Lanthanum is transported by the sodium/calcium exchanger and regulates its activity.

La3+ uptake was measured in fura 2-loaded Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger (NCX1.1). La3+ was taken up by the cells after an initial lag phase of 50-60 s and achieved a steady state within 5-6 min.

Cellular regulation of sodium-calcium exchange.

Na(+)/Ca(2+) exchange activity was studied in transfected Chinese hamster ovary (CHO) cells expressing the wild-type cardiac exchanger (NCX1.1) or mutants created by site-directed mutagenesis. The activity of the wild-type exchanger, but not exchanger mutants deficient in Ca(2+)-dependent activation, was inhibited by sphingolipids such as ceramide and sphingosine.