Abstract
Chinese hamster ovary cells expressing the bovine cardiac Na/Ca exchanger were treated with ouabain to increase [Na+]i and stimulate Ca2+ influx by Na/Ca exchange. Depletion of cellular ATP inhibited 45Ca uptake by 40% or more and reduced the half-maximal Na+ concentration for inhibition of 45Ca uptake from 90 to 55 mM. ATP depletion also reduced the rate of rise in [Ca2+]i when [Na+]o was reduced and inhibited the decline in [Ca2+]i when high [Na+]o was restored. The effects of ATP depletion were either absent or reduced in cells expressing a mutant exchanger missing most of the cytosolic hydrophilic domain. We were unable to detect a phosphorylated form of the exchanger in immunoprecipitates from 32P-labeled cells. ATP depletion caused a breakdown in the actin cytoskeleton of the cells. Treatment of the cells with cytochalasin D mimicked the effects of ATP depletion on the [Na+] inhibition profile for 45Ca uptake. Thus, ATP depletion inhibits both the Ca2+ influx and Ca2+ efflux modes of Na/Ca exchange, and may alter the competitive interactions of extracellular Na+ and Ca2+ with the transporter. The latter effect appears to be related to changes in the actin cytoskeleton.
Highlights
Chinese hamster ovary cells expressing the bovine cardiac Na/Ca exchanger were treated with ouabain to increase [Na"], and stimulate Ca2+ influx by Na/Ca exchange
Cells-CK1.4 cells were prepared by transfection of dhfr :? Chinese hamster ovary cells with a mammalian expression vector containing a cDNA insert coding for the bovine cardiac Na/Ca exchanger [16]
ATP Depletion and 45Ca Uptake in CK1.4 Cells-To determine the effects of ATP depletion on Na/Ca exchange activity, CK1.4 cells were preincubated for 30 min in the absence of glucose and treated with a combination of mitochondrial inhibitors: rotenone (2 /LM) and oligomycin (2.5 /Lg/ml)
Summary
Vol 270, No 16, Issue of April 21, pp. 9137-9146, 1995 Printed in U.S.A. ATP-dependent Regulation of Sodium-Calcium Exchange in Chinese Hamster Ovary Cells Transfected with the Bovine Cardiac SodiumCalcium Exchanger*. ATP depletion strongly inhibits the Ca2 + efflux mode of Na/Ca exchange and potentiates the inhibitory effect of extracellular Na" on Ca2 + influx These effects are absent or reduced in cells expressing the deletion mutant. This suggests that ATP-dependent regulation, like secondary activation by cytosolic Ca2 + , is mediated by regions within the hydrophilic domain of the exchanger. Portions of these results were presented previously in abstract form [17,18]
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