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) f orm large aqueous pores whose walls are believed to consist of 1 alpha helix and 12 beta strands. Each channel has two voltage-gating proces ses: one closes the channels at positive potentials, the other at nega tive. When VDAC is reconstituted into phospholipid (soybean) membranes , the two gating processes have virtually the same steepness of voltag e dependence and the same midpoint voltage. Substituting lysine for gl utamate at either end of one putative beta strand (E145K or E152K) mad e the channels behave asymmetrically, increasing the voltage dependenc e of one gating process but not the other. The asymmetry was the same whether 1 or 100 channels were in the membrane, indicating oriented ch annel insertion. However, the direction of insertion varied from membr ane to membrane, indicating that the insertion of the first channel wa s random and subsequent insertions were directed by the previously ins erted channel(s), This raises the prospect of an auto-directed inserti on with possible implications to protein targeting in cells. Each of t he mutations affected a different gating process because the double mu tant 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 w here it may act as a tether and guide/restrict the motion of the senso r.