I'm not sure I quite understand.....if you use a PNG file there will be some transparency already built in, and whatever you play on a layer "behind" this will only show through in those transparent spaces.....when you build the PNG your "positive" space will actually be black to simulate the spaces you would have between tubes with a real Versa-Tube rig.
Another option woudl be to build a B&W JPEG and use the "Transparent Whites" or "Transparent Blacks" color FX to create a transparent area.