Membrane perturbations induced by the interactions of zinc ions with band 3 in human erythrocytes

Abstract

Of group 12 metals, zinc is an essential element to maintain our life, but other metals such as cadmium and mercury are toxic in cellular activities. Interactions of these metals with biomembranes are important to understand their effects on our living cells. Here, we describe the membrane perturbations induced by these metals in human erythrocytes. Of these metals, Zn²⁺ ions only induced the erythrocyte agglutination. Histidine residues in extracellular domains of band 3 participated in Zn²⁺-induced agglutination. Interestingly, it was found that band 3-cytoskeleton interactions play an important role in Zn²⁺-induced agglutination. In contrast with Hg²⁺ and Cd²⁺ ions, Zn²⁺ ions greatly suppressed pressure-induced hemolysis by cell agglutination. Such a suppression was removed upon dissociation of agglutinated erythrocytes by washing, indicating the reversible interactions of Zn²⁺ ions with erythrocyte membranes. Excimer fluorescence of pyrene indicated that spectrin is denatured by a pressure of 200 MPa irrespective of hemolysis suppression. Taken together, these results suggest that the agglutination of erythrocytes due to the interactions of Zn²⁺ ions with band 3 is stable under pressure, but spectrin, cytoskeletal protein, is denatured by pressure.

Keywords: 5P8, 5 mM sodium phosphate, pH 8; Agglutination; Band 3; C12E8, octaethylene glycol mono-n-dodecyl ether; DEPC, diethylpyrocarbonate; DIDS, 4,4′-diisothiocyanostilbene- 2,2′-disulfonate; DMPC, dimyristoylphosphatidylcholine; DNDS, 4, 4′-dinitrostilbene- 2,2′-disulfonate; Erythrocyte; Excimer; Hemolysis; NPIA, N-(1-pyrenyl) iodoacetamide; PBS, phosphate-buffered saline, 10 mM sodium phosphate, 150 mM NaCl, pH 7.4; SEM, scanning electron microscope; TBS, tris-buffered saline, 17 mM Tris, 123 mM NaCl, pH 7.4; TM, transmembrane; Zinc.

Graphical abstract

Fig. 1

Erythrocyte agglutination by Zn²⁺ions. Erythrocytes in TBS (A) or 4 mM Zn²⁺(B), 4 mM Cd²⁺(C), 10 μM Hg²⁺(D)-containing TBS were incubated for 10 min at 37 °C and observed under a light microscope.

Fig. 2

Interactions of band 3 with Zn²⁺ ions. A, SDS-PAGE of band 3-rich vesicles. Erythrocytes in PBS containing DMPC liposomes were incubated for 5 h at 37 °C. Lane 1, ghosts from intact erythrocytes; lane 2, ghosts from DMPC-treated erythrocytes; lane 3, vesicles from DMPC-treated erythrocytes. B, Size of Zn²⁺-treated vesicles. Vesicles from DMPC-treated erythrocytes were suspended in TBS containing 0.5 mM Zn²⁺ ions. C, DIDS effects on Zn²⁺-induced agglutination of erythrocytes. Intact erythrocytes or 50 μm DIDS-treated ones were suspended in TBS containing 0.5 mM Zn²⁺ ions and turbidity of suspensions was monitored at 700 nm. A₀ and A_t are absorbance at 0 and t min of erythrocyte suspensions, respectively. Times (t min) corresponding to A_t /A₀=0.5 are shown in right panel. All values are means±SD for three independent experiments.

Fig. 3

Interactions of Zn²⁺ ions with histidines on band 3. A, Dispersed or agglutinated erythrocytes on agglutination plate. Left well, cells suspended in TBS; middle well, cells suspended in TBS containing 0.5 mM Zn²⁺ ions; right well, Zn²⁺-treated cells suspended in TBS containing 0.5 mM histidine. B, DEPC effects on Zn²⁺-induced agglutination. Intact erythrocytes or 7 mM DEPC- treated ones were suspended in TBS containing 0.5 mM Zn²⁺ ions. C, Effects of pH on Zn²⁺-induced agglutination. Erythrocytes were suspended in TBS (pH 5.8–8.6) containing 0.5 mM Zn²⁺ ions. D, Band 3-cytoskeleton interactions. Eosin-5-maleimide-labeled ghosts were solubilized by 0.5% C12E8 in TBS (pH 7.4 and 8.6) and centrifuged. All values are means±SD for three independent experiments. ^*p<0.03 vs. pH 7.4.

Fig. 4

Effects of group 12 metal ions on pressure-induced hemolysis. A, Erythrocytes were suspended in TBS containing 4 mM Zn²⁺, 4 mM Cd²⁺, or 10 μm Hg²⁺ ions and exposed to a pressure of 200 MPa in the presence of metal ions. B, Erythrocytes pretreated with metal ions in A were washed with TBS and compressed in the absence of metal ions. All values are means±SD for three independent experiments. ^*p<0.05 vs. none.

Fig. 5

Membrane properties at 200 MPa of erythrocytes agglutinated by 0.5 mM Zn²⁺ ions. A, SEM of pressure-treated erythrocytes. Left panel, intact cells; right panel, agglutinated cells were compressed and dissociated by 1 mM EDTA. B, Excimer fluorescence appears upon compression of agglutinated erythrocytes. Agglutinated cells were compressed, dissociated using EDTA, and used for ghost preparation. Resealed ghosts were labeled with NPIA. F₃₈₈ and F₄₇₀ are fluorescence intensities at 388 and 470 nm, respectively. All values are means±SD for three independent experiments.