Oriented channel insertion reveals the motion of a transmembrane beta strand during voltage gating of VDAC.

Yeast VDAC channels (isolated from the mitochondrial outer membrane) form large aqueous pores whose walls are believed to consist of 1 alpha helix and 12 beta strands. Each channel has two voltage-gating processes: one closes the channels at positive potentials, the other at negative. When VDAC is reconstituted into phospholipid (soybean) membranes, the two gating processes have virtually the same steepness of voltage dependence and the same midpoint voltage. Substituting lysine for glutamate at either end of one putative beta strand (E145K or E152K) made the channels behave asymmetrically, increasing the voltage dependence of one gating process but not the other. The asymmetry was the same whether 1 or 100 channels were in the membrane, indicating oriented channel insertion. However, the direction of insertion varied from membrane to membrane, indicating that the insertion of the first channel was random and subsequent insertions were directed by the previously inserted channel(s). This raises the prospect of an auto-directed insertion with possible implications to protein targeting in cells. Each of the mutations affected a different gating process because the double mutant increased voltage dependence of both processes. Thus this strand may slide through the membrane in one direction or the other depending on the gating process. We propose that the model of folding for VDAC be altered to move this strand into the sensor region of the protein where it may act as a tether and guide/restrict the motion of the sensor.