Passive transport pathways for Ca2+ and Co2+ in human red blood cells. 57Co2+ as a tracer for Ca2+ influx

The passive transport of calcium and cobalt and their interference were studied in human red cells using Ca-45 and Co-57 as tracers. In ATP-depleted cells, with the ATP concentration reduced to about 1 mu M, the progress curve for Ca-45 uptake at 1 mM rapidly levels off with time, consistent with a residual Ca-pump activity building up at increasing [Ca-T](c) to reach at [Ca-T](c) about 5 mu mol (1 cells)(-1) a maximal pump rate that nearly countermands the passive Ca influx, resulting in a linear net uptake at a low level. In ATP-depleted cells treated with vanadate, supposed to cause Ca-pump arrest, a residual pump activity is still present at high [Ca-T](c). Moreover, vanadate markedly increases the passive Ca2+ influx. The residual Ca-pump activity in ATP-depleted cells is fuelled by breakdown of the large 2,3-DPG pool, rate-limited by the sustainable ATP-turnover at about 40-50 mu mol (I cells)(-1) h(-1). The apparent Ca2+ affinity of the Ca-pump appears to be markedly reduced compared to fed cells. The 2,3-DPG breakdown can be prevented by inhibition of the 2,3-DPG phosphatase by tetrathionate, and under these conditions the 45Ca uptake is markedly increased and linear with time, with the unidirectional Ca influx at 1 mM Ca2+ estimated at 50-60 mu mol (I cells)(-1) h(-1). The Ca influx increases with the extracellular Ca2+ concentration with a saturating component, with K-1/2(Ca) about 0.3 mM, plus a non-saturating component. From Ca-45-loaded, ATP-depleted cells the residual Ca-pump can also be detected as a vanadate- and tetrathionate-sensitive efflux. The Ca-45 efflux is markedly accelerated by external Ca2+, both in control cells and in the presence of vanadate or tetrathionate, suggesting efflux by carrier-mediated Ca/Ca exchange. The Co-57 uptake is similar in fed cells and in ATP-depleted cells (exposed to iodoacetamide). consistent with the notion that Co2+ is not transported by the Ca-pump. The transporter is thus neither - SH-group nor ATP or phosphorylation dependent. The Co-57 uptake shows several similarities with the Ca-45 uptake in ATP-depleted cells supplemented with tetrathionate. The uptake is linear with time, and increases with the cobalt concentration with a saturating component, with J(max) about 16 mu mol (I cells)(-1) h(-1) and K-1/2(Co) about 0.1 mM, plus a non-saturating component The Co-57 and Ca-45 uptake shows mutual inhibition, and at least the stochastic Ca2+ influx is inhibited by Co2+. The Co-57 and Ca-45 uptake are both insensitive to the 1,4-dihydropyridine Ca-channel blocker nifedipine, even at 100 mu M. The Co-57 uptake is increased at high negative membrane potentials, indicating that the uptake is at least partially electrogenic. The Co-57 influx amounts to about half the Ca-45 influx in ATP-depleted cells. It is speculated that the basal Ca2+ and Co2+ uptake could be mediated by a common transporter, probably with a channel-like and a carrier-mediated component, and that Co-57 could be useful as a tracer for at least the channel-like Ca2+ entry pathway in red cells, since it is not itself transported by the Ca-pump and, moreover, is effectively buffered in the ctosol by binding to hemoglobin, without interfering with Ca2+ buffering. The molecular identity of the putative common transporter(s) remains to be defined. (C) 2011 Elsevier Inc. All rights reserved.