Voltage-dependent Anion Channel-1 (VDAC-1) Contributes to ATP Release and Cell Volume Regulation in Murine Cells

Abstract

Extracellular ATP regulates several elements of the mucus clearance process important for pulmonary host defense. However, the mechanisms mediating ATP release onto airway surfaces remain unknown. Mitochondrial voltage-dependent anion channels (mt-VDACs) translocate a variety of metabolites, including ATP and ADP, across the mitochondrial outer membrane, and a plasmalemmal splice variant (pl-VDAC-1) has been proposed to mediate ATP translocation across the plasma membrane. We tested the involvement of VDAC-1 in ATP release in a series of studies in murine cells. First, the full-length coding sequence was cloned from a mouse airway epithelial cell line (MTE7b−) and transfected into NIH 3T3 cells, and pl-VDAC-1-transfected cells exhibited higher rates of ATP release in response to medium change compared with mock-transfected cells. Second, ATP release was compared in cells isolated from VDAC-1 knockout [VDAC-1 (−/−)] and wild-type (WT) mice. Fibroblasts from VDAC-1 (−/−) mice released less ATP than WT mice in response to a medium change. Well-differentiated cultures from nasal and tracheal epithelia of VDAC-1 (−/−) mice exhibited less ATP release in response to luminal hypotonic challenge than WT mice. Confocal microscopy studies revealed that cell volume acutely increased in airway epithelia from both VDAC-1 (−/−) and WT mice after luminal hypotonic challenge, but VDAC-1 (−/−) cells exhibited a slower regulatory volume decrease (RVD) than WT cells. Addition of ATP or apyrase to the luminal surface of VDAC-1 (−/−) or WT cultures with hypotonic challenge produced similar initial cell height responses and RVD kinetics in both cell types, suggesting that involvement of VDAC-1 in RVD is through ATP release. Taken together, these studies suggest that VDAC-1, directly or indirectly, contributes to ATP release from murine cells. However, the observation that VDAC-1 knockout cells released a significant amount of ATP suggests that other molecules also play a role in this function.

ATP release from non-excitable cells

“…VDAC is a mitochondrial ATPpermeable membrane pore, but has been suggested also to be present in the plasma membrane as a specific splice variant (pl-VDAC-1) [72]. Interestingly, VADC-1 knockout mouse tissue shows reduced mechanically stimulated ATP release and, vice versa, overexpression of pl-VDAC-1 protein in fibroblasts leads to increased mechanically stimulated ATP release [73]. Thus, these data support a role of this protein in ATP release.…”

“…Thus, these data support a role of this protein in ATP release. However, mechanically triggered ATP release continues to be present in the absence of pl-VDAC-1 [73]. Another conflicting finding regarding VDAC is its very long lag time before activation of the membrane current (>10 min) [70], a finding which argues against its involvement in swelling-induced ATP release that always occurs promptly [67].…”

Vesicular and conductive mechanisms of nucleotide release

“…The multidrug resistance (MDR)-1 protein, the CF transmembrane conductance regulator (CFTR) Cl − channel, and the mitochondrial voltage-dependent anion channel-1 (VDAC-1) were initially proposed as ATP release pathways or facilitators of ATP release in various cell types, but the involvement of these proteins in an ATP release function could not be corroborated by a number of studies [90][91][92][93][94][95][96]; for a critical review on these proposed pathways, see [8,97]. More recently, two classes of plasma membrane channels have been associated with an ATP conductive activity: (1) Cl − channels such as maxi anion channels, volume-regulated ion channels, and tweety; and (2) pore forming connexins, pannexins, and P2X7 receptors.…”

ATP as a mediator of macula densa cell signalling

“…We found that this was a Cl − -permeable pathway since the I-V relationship was unaltered when N-methyl-D-glucamine was substituted for Na + and inward currents were reduced and the reversal potential was shifted to the left when bath [Cl − ] was lowered by substituting with gluconate. The maxi-anion channel was permeable to large monovalent anions, and previous studies had found that this channel was permeable to ATP [112]. To assess whether the macula densa basolateral membrane channel was ATP-permeable, the intracellular (bath) solution was changed to 100 mM ATP solution (Fig.…”

“…Although the physical presence of a channel that conducts ATP has been firmly established using biophysical techniques, the molecular nature of this maxi-anion channel has remained elusive. Based on the biophysical properties of this channel, it has been suggested that it is a plasma membrane form of a mitochondrial porin (voltagedependent anion channel, VDAC) [112]. This is especially appealing, since it is well known that this channel transports ATP across mitochondrial membranes.…”