Richard W. Hendershot and Professor David Busath, Zoology
Botulinum toxin is an exotoxin produced by the bacterium Clostridium botulinum and is the cause of botulism poisoning in poorly canned food. It is known that the toxin consists of two parts namely the larger Heavy Chain and the smaller Light Chain. Previous data suggests that the larger Heavy Chain forms channels in the cell membrane facilitating infection of the cell by the smaller Light Chain 1. It is also known that the Heavy Chain does indeed form channels in a bilayer system but channels formed by a single Heavy Chain are too small for the light chain to pass through. The purpose of my project was to study the characteristics of channels formed by the Heavy Chain in hopes of gaining a greater understanding of the intoxication process.
A lipid bilayer was formed through the use of Diphytenoyl phosphatidal choline (DPhPC) in octane at a concentration of 20 mg/ml. The DPhPC solution was spread over a 150 μm hole. The bilayer formed had a measured capacitance of 40-80 ‘F and a 100 mV potential was applied to the cis side. The experiment took place in a 1M potassium chloride solution.
I attempted to facilitate the insertion of the botulinum toxin by inserting a high concentration of toxin into the cis chamber. The attempt was unsuccessful and for several months I continued to attempt channel formation by varying lipids from DPHPC to PE and PS both commonly used lipids. I continued to be unsuccessful save for one probable success which is included, I hypothesized that the membrane was simply too thick —inflated by the either the lipid (DPHPC) or the solvent (octane). But simply changing the two had little effect. I then hypothesized that the geometry of the cuvette was inconsistent for the type of bilayer I was attempting to form and upon using a different setup the acquired bilayer had a much higher capacitance (180-250 pF indicating a much thinner bilayer). I plan further experiments to test the validity of my hypothesis but as of yet have been unable to do so.
While in the process of seeking to discover the source of my failure to successfully form botulinum neurotoxin protein channels I often used one of the oldest known protein channels —gramicidin A to check my technique. In the process of doing so I discovered an unusual behavior as yet observed. Generally gramicidin A forms channels in a lipid bilayer at low concentrations in the order of 10-6 mg/ml. This was true of the experiments I ran using the gramicidin protein. Occasionally however what I termed “mystery channels” would appear. Normally gramicidin channels have a channel period from 0.5 to 4 seconds in length with a current around 3 pA with a 100 mV applied potential. The mystery channels however, stayed open for at least twenty minutes with a 100 mV applied potential allowing a 7- 10 pA current to pass through. Much larger and longer lasting than any type of gramicidin channel previously observed. At first I suspected that these channels might be artifacts of the amplifier but after the appearance of these mystery channels became fairly reproducible that option was ruled out. I also wondered about contamination of the experimental set-up being used and that too was ruled out after a very thorough cleaning failed to eliminate the sporadic appearance of the “mystery channels.” Some parameters to the appearance of these channels has been observed: they have only appeared when DPHPC has been used in octane, the bilayer capacitance has measured greater than 60 pF and a small concentration of gramicidin is inserted into the chamber.
I originally set out to discover the channel-forming properties of the botulinum neurotoxin but because of my lack of success I possibly discovered a previously unknown conformer of the gramicidin protein. I plan to spend much of this upcoming year in hopes to formulate a database in order to later publish the results of this year’s experiments. I will also continue to attempt to successfully form botulinum protein channels with the aid of a second student and to test the hypothesis that the lack of channel formation is due to bilayer thickness.
References
- M.F,Schmid, 1993. Nature 364: 827-830.
