VDAC closure increases calcium ion flux

Abstract

VDAC is the major permeability pathway in the mitochondrial outer membrane and can control the flow of metabolites and ions. Therefore Ca(2+) flux across the outer membrane occurs mainly through VDAC. Since both Ca(2+) fluxes and VDAC are involved in apoptosis, we examined whether Ca(2+) is required for channel formation by VDAC isolated from rat liver. The voltage gating of VDAC does not require Ca(2+) and it functions normally with or without Ca(2+). Additionally, VDAC generally shows a higher permeability to Ca(2+) in the closed states (states with lower permeability to metabolites) than that in the open state. Thus VDAC closure, which induces apoptosis, also favors Ca(2+) flux into mitochondria, which can also lead to permeability transition and cell death. These results are consistent with the view that VDAC closure is a pro-apoptotic signal.

Fig. 1

VDAC was reconstituted into a planar phospholipid membrane composed of 0.5% PC, 0.5% asolectin and 0.05% cholesterol in the presence of 1.0 M KCl, 20mM HEPES, pH=7.2. 0.4mM EGTA was then added to each side of the chamber to chelate any possible trace amount of Ca²⁺. 2mM Ca²⁺ was added later to compare VDAC’s gating in the presence and absence of Ca²⁺. (Note: there is enough buffer to control the pH even after acid is released following chelation of some Ca²⁺) VDAC’s gating was recorded by applying 50 mV (cis side is ground). The total conductance in the absence and presence of Ca²⁺ is 115 nS and 179 nS, respectively. There is an 18-minute gap between these records and the conductance increased because of VDAC insertion. The percent conductance drop and the rate of decay are essentially the same with and without free Ca²⁺.

Fig. 2

An example of fitting equation 2 and equation 3 to a segment of a single-channel current-voltage record representing one state of a VDAC channel (a closed state here). A four-fold gradient of CaCl₂ was present (see Materials and Methods). The arrows and associated numbers indicate the parameters obtained from the fitting.

Fig. 3

Permeability of VDAC to calcium and chloride ions as a function of the total conductance of the channel. Each value is obtained directly as in Fig. 2.

Fig. 4

Comparison of the permeability of Ca²⁺ through VDAC in different states. The values are mean ± S.E. of 15 measurements in the open state and the negatively closed states and 19 measurements in the positively closed states. (*P < 0.05, *** P < 0.001, compared to the open state)

Fig. 5

(A) The open probability of VDAC in response to voltage. (B) A comparison of the average calcium flux through a single VDAC channel in response to voltage calculated for what it would be in the presence of 1 µM cytosolic Ca²⁺ (0 µM in the intermembrane space). The filled circles are the weighted average flux of Ca²⁺ through the open and closed states, assuming the same voltage gating property as in panel A. The open circles are assuming that VDAC is always in the open state.