Escherichia coli alpha-hemolysin triggers shrinkage of erythrocytes via K(Ca)3.1 and TMEM16A channels with subsequent phosphatidylserine exposure

Abstract

alpha-Hemolysin from Escherichia coli (HlyA) readily lyse erythrocytes from various species. We have recently demonstrated that this pore-forming toxin provokes distinct shrinkage and crenation before it finally leads to swelling and lysis of erythrocytes. The present study documents the underlying mechanism for this severe volume reduction. We show that HlyA-induced shrinkage and crenation of human erythrocytes occur subsequent to a significant rise in [Ca(2+)](i). The Ca(2+)-activated K(+) channel K(Ca)3.1 (or Gardos channel) is essential for the initial shrinkage, because both clotrimazole and TRAM-34 prevent the shrinkage and potentiate hemolysis produced by HlyA. Notably, the recently described Ca(2+)-activated Cl(-) channel TMEM16A contributes substantially to HlyA-induced cell volume reduction. Erythrocytes isolated from TMEM16A(-/-) mice showed significantly attenuated crenation and increased lysis compared with controls. Additionally, we found that HlyA leads to acute exposure of phosphatidylserine in the outer leaflet of the plasma membrane. This exposure was considerably reduced by K(Ca)3.1 antagonists. In conclusion, this study shows that HlyA triggers acute erythrocyte shrinkage, which depends on Ca(2+)-activated efflux of K(+) via K(Ca)3.1 and Cl(-) via TMEM16A, with subsequent phosphatidylserine exposure. This mechanism might potentially allow HlyA-damaged erythrocytes to be removed from the bloodstream by macrophages and thereby reduce the risk of intravascular hemolysis.

FIGURE 1.

Volume changes in human erythrocytes induced by HlyA from E. coli. A, representative DIC images of erythrocytes before and after HlyA stimulation (∼25 ng ml⁻¹). B and C, representative traces showing side scatter (B) and forward scatter (C) over time (0–45 min) after HlyA (3 ng ml⁻¹) is applied. Each dot represents a single erythrocyte. The values on the y axis are arbitrary. D and E, mean normalized values of side scatter (D) and forward scatter (E) over time (0–45 min) after HlyA (3 ng ml⁻¹) is applied (open squares) and time controls (filled circles). F, side scatter as a function of forward scatter at four time points (0, 5, 10, and 45 min) after HlyA is added (3 ng ml⁻¹). Each dot represents a single erythrocyte. The values on the axis are arbitrary and shown as the means ± S.E. (n = 8).

FIGURE 2.

Effect of HlyA on [Ca²⁺]_i in human erythrocytes. A, number of cells as a function of fluo 4 fluorescence signal shows two populations of cells at time 0, 2, 6, and 10 min after HlyA is applied (3 ng ml⁻¹). The population of cells with a substantial rise in [Ca²⁺]_i (as indicated by the red bars) is defined as [Ca²⁺]_i increasing cells. B, side scatter as a function of forward scatter at time 0, 2, 6, and 10 min after HlyA is applied (3 ng ml⁻¹). The cells marked in red (the same population as marked with the red bar in A show a rise in [Ca²⁺]_i. Each dot represents a single erythrocyte. The values on the axis are arbitrary. C, the corresponding effect of the Ca²⁺ ionophore, ionomycin. At the left the numbers of cells are shown as a function of fluo 4 fluorescence signal. Again during control there are two populations of cells, one with low and a smaller with higher [Ca²⁺]_i. Only a single population of cells with high [Ca²⁺]_i was observed after ionomycin was applied (1 μm, 10 min). At the right the side scatter is shown as a function of forward scatter during control, and 10 min after ionomycin was applied. The cells marked in red (the same population as marked with a red bar in the left panels) show a rise in [Ca²⁺]_i. Each dot represents a single erythrocyte. The values on the axis are arbitrary. D, the percentage of erythrocytes with a rise in [Ca²⁺]_i in response to HlyA. The values are the means ± S.E. (n = 5). E, the mean values of cells with a rise in [Ca²⁺]_i after addition of ionomycin (1 μm). The values are the means ± S.E. (n = 5). F, the traces show the change in fluo 4 fluorescence over time in 23 erythrocytes in response to HlyA. G, imaging of fluo 4-loaded human erythrocytes exposed to HlyA (25 ng ml⁻¹).

FIGURE 3.

The effect of K_Ca1. 3 channel inhibitors on HlyA-induced volume changes and lysis in human erythrocytes. A, the effect of clotrimazole (CLT, 20 μm, filled circles) on the changes in forward scatter induced by HlyA (3 ng ml⁻¹) compared with controls (open squares). B, shows the effect of TRAM-34 (10 μm, filled triangles) on the change in forward scatter induced by HlyA (3 ng ml⁻¹) compared with controls (open squares). C and D, the corresponding changes in side scatter in response to clotrimazole and TRAM-34 are shown in C and D, respectively. All of the above values are normalized to time 0 and given as mean values ± S.E. (n = 5–6). The hemolysis was measured as the level of released hemoglobin in the absence or presence of increasing concentrations of clotrimazole (E) and TRAM-34 (F) at two concentrations of HlyA leading to 20 and 50% lysis after 60 min (5 and 10 ng ml⁻¹, respectively). The values are given as the means ± S.E. (n = 4–6).

FIGURE 4.

HlyA-induced crenation and lysis in erythrocytes from TMEM16A^−/−versus TMEM16A^+/+ mice. A, the number of erythrocytes isolated from TMEM16A^+/+ and TMEM16A^−/− mice, which crenate in response to HlyA (25 ng ml⁻¹). B, the subsequent lysis of erythrocytes isolated from TMEM16A^+/+ and TMEM16A^−/− mice in response to HlyA. The values are given as the means ± S.E. (n = 8 for TMEM16A^+/+ mice and n = 6 for TMEM16A^−/− mice). The asterisks indicate statistically significance between the values obtained from TMEM16A^−/− and TMEM16A^+/+ mice.

FIGURE 5.

HlyA induced PS exposure (annexin V binding) in human erythrocytes is inhibited by K_Ca1. 3 channel antagonists. A and B, representative experiment showing side scatter as a function of forward scatter during control (A) and after HlyA (1 ng ml⁻¹) is applied (B). The HlyA concentration (1 ng ml⁻¹) was adjusted to completely avoid hemolysis during the observation period. The circled regions in A and B define shrunken cells. C, mean value of erythrocyte shrinkage after HlyA is applied (n = 10). Ionomycin (1 μm) was used as a positive control for cell shrinkage (n = 5). D and E, annexin V binding in the same cells as shown in A and B during control (D) and after HlyA is applied (E). The boxed regions define cells which are both shrunken and positive for annexin V staining. F, mean value of number of cells binding annexin V (n = 10). Ionomycin (1 μm) was used a positive control for annexin V binding (n = 5). G, K_Ca1.3 channel antagonists reduce HlyA-induced PS exposure in human erythrocytes. Erythrocytes incubated with annexin V during control and HlyA application (1 ng ml⁻¹) in the absence or presence of either TRAM-34 (1 and 10 μm) or clotrimazole (CLT, 1 and 5 μm). The values are given as the means ± S.E. (n = 5–7). The asterisks indicate statistical significance.