Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

Abstract

A systematic study was made of the action of 4-acet-amido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on active Ca2+ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca2+-selective electrodes or use of tracer 45Ca2+. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS, Ca2+ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K+ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca2+ pump, exchanging one Ca2+ ion for one proton.

Probing the extracellular release site of the plasma membrane calcium pump

“…1 and 3 are both fit with a hyperbolic curve, i.e., a Hill coefficient of 1. The stoichiometry of PMCA has been reported to be 1 Ca 2ϩ -2 H ϩ and 1 Ca 2ϩ -1 H ϩ (11,19,21,25,26,31), and our activation curves are consistent with 1 H ϩ activating. However, although our data do not require a model with 2 H ϩ activating, our data also do not eliminate such a model.…”

“…The Ca 2ϩ pump not only mediates Ca 2ϩ efflux, but also proton (H ϩ ) influx. The H ϩ influx in red blood cells was initially inferred from indirect experiments (6,21,25,26,31). We have used a pH stat technique to measure directly Ca 2ϩ /H ϩ exchange in intact red blood cells (19).…”

Reversible inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid of the plasma membrane calcium-magnesium ATPase from kidney proximal tubules

“…The negatively charged amino-reactive reagent DIDS and its dihydro derivative H2DIDS have been widely used in the study of both anion exchange (Zaki et al, 1975;Lepke et al, 1976;Cabantchik et al, 1978;Kasai & Taguchi, 1981) and the relationship between anion fluxes and those of other ionic species, including Ca2+ (Campbell & MacLennan, 1980;Waisman et al, 1981Waisman et al, , 1982Romero & Ortiz, 1988). However, these compounds appear to operate in different systems in a way that is not directly related to anion translocation (Campbell & MacLennan, 1980;Niggli et al, 1982; Waisman et al, 1982;Kimura et al, 1988;Pedemonte & Kaplan, 1988).…”

The Cation Selectivity of the ZIP Transporters

“…mZIP14 can transport zinc as well as iron (II) (Liuzzi, Aydemir, Nam, Knutson, & Cousins, 2006). However, the mechanism of inhibition by DIDS for other proteins, such as the Ca 2þ -ATPase, is not well understood (Romero & Ortiz, 1988;Waisman, Smallwood, Lafreniere, & Rasmussen, 1982). Interestingly, it was observed that labeling with 4,4 0 -diisothiocyanatostilbene-2,2 0 -disulfonic acid (DIDS), a lysine-reactive probe, reduced 109 Cd 2þ uptake in a concentration-dependent manner.…”