Inhibition of the red blood cell calcium pump by eosin and other fluorescein analogues

Abstract

This paper addresses the mechanism of inhibition of the plasma membrane Ca pump by fluorescein analogues and their isothiocyanate derivatives. Eosin (i.e., tetrabromofluorescein) was found to be one of the most potent reversible inhibitors of the erythrocyte Ca pump [half-maximal inhibitory concentration (IC50) < 0.2 microM]; fluorescein itself was about four orders of magnitude less potent (IC50 approximately 1,000 microM). Eosin decreased the maximum influx and thus did not compete with ATP for the Ca pump. Irreversible inhibition produced by the isothiocyanate analogues of eosin and fluorescein [eosin 5-isothiocyanate (EITC) and fluorescein 5-isothiocyanate (FITC), respectively] was also studied. While EITC bound reversibly at the eosin site, two results suggest that EITC does not react covalently at this site: 1) eosin did not alter the time course of the EITC irreversible reaction, and 2) the concentration dependence for reversible EITC inhibition was different from the concentration dependence for irreversible EITC inhibition. ATP did slow the rate of inactivation of both EITC and FITC consistent with the idea that EITC and FITC bind to the ATP site. Our results are consistent with eosin and ATP binding to separate sites and EITC reacting covalently at the ATP site, but not the eosin site.

Kinetic characterization of Na,K-ATPase inhibition by Eosin

“…Eosin, in addition to inhibiting the Na pump, also inhibits the plasma membrane Ca pump from red cells [20] and smooth muscle [35]. Interestingly, those studies found that eosin was not competitive with ATP, similar to the results presented here for the Na pump.…”

“…Indeed, the nucleotide site binding efficiency of eosin remains an exploited tool for real-time fluorescent monitoring of nucleotide binding site conformational changes [17,18,19]. Thus, it was surprising when we observed that eosin non-competitively inhibited ATP hydrolysis and ATP-dependent 45 Ca ++ transport in the red cell plasma membrane Ca-ATPase [20]. Yet, like other P-type ATPases, FITC was an irreversible, ATP-protectable, inhibitor of PMCA, which modified the same conserved lysine residue (i.e.…”

“…Purified Na,K-ATPase was obtained as described previously [22]. Red cell membranes were prepared as described previously [20]. Protein concentrations were determined by the method of Lowry et al [23].…”

“…Red Cell Na,K-ATPase activity was measured as reported previously for the red cell Ca pump [20], with minor modifications for Na,K-ATPase. Briefly, 0.5 mg of red cell membrane protein was diluted into 30 mL of assay media containing (in mM): 140 NaCl, 34 KCl, 33 HEPES (pH = 7.4), 4 MgCl 2 , and 0.06 EGTA.…”

Demonstrations

“…However, they can lack resolution, can be difficult to calibrate and may be toxic. BCECF also inhibits the Ca 2 + -H+ pump (Gatto & Milanick, 1993), which is an important activity-dependent acid loader in neurones (Trapp et al 1996;Schwiening, 1997).…”