Probing the Nature of the Voltage-Sensing Mechanism of the Mitochondrial Outer Membrane Channel, VDAC: Initial Kinetic Analysis and Aluminum Chloride-Induced Alterations

Abstract

VDAC channels are the major permeability pathways through the mitochondrial outer membrane. They exist in a high conducting, "open," state at low potentials and a low conducting, "closed," state at high potentials (>+/- 20mV). The mechanism underlying voltage-dependent behavior is poorly understood. VDAC isolated from Neurospora crassa were studied in planar phospholipid membranes. Aluminum chloride interaction with the protein and analysis of the rates of channel opening and closing were used to probe the mechanism of voltage-dependence. Micromolar amounts of aluminum chloride (>1 uM) decreased the steepness of the voltage-dependence and increased the voltage needed to close half the channels. Open and closed channel conductance levels were essentially unchanged. Neither channel conformation nor ion selectivity were altered. The effective aluminum species is either, or both, aluminate or aluminum hydroxide. The rate constants of channel opening and closing were determined from multi-channel membrane studies. Closure rate constants increased exponentially with increased negative applied voltages, from 0.01/sec at -30 mV to 1.77/sec at -80 mV. Short periods (< 4-6 min) in the open state before closure decreased closure rates, indicating the presence of at least two open states. Opening rates were at least an order of magniture faster than closure rates and had no marked voltage-dependence between -15 mV and -5 mV or +5 mV and +10 mV. Channel closure accounts for 80%, or more, of the voltage-dependence observed in the steady state. Implications of these findings for modeling the action of the channel are discussed. As a result of these studies future models of the VDAC channel and research must consider these new complexities: distinct groups of charges are responsible for voltage-gating and selectivity; the sensor is likely outside the channel proper; channel opening may involve a large dipole; and opening and closing may occur via different molecular pathways.