Tetanus Toxin Forms Channels in Planar Lipid Bilayers Containing Gangliosides

Abstract

Although the agent responsible for the spasticity and convulsions characteristic of human tetanus was identifed at the turn of the century as the neurotoxin produced by the bacterium Clostridium tetani (1), the molecular basis for the action of the toxin at the central nervous system remains obscure (1, 2). Tetanus toxin (TT), a soluble protein of M, -150,000 in its native form, has no enzymatic activity identified thus far. It is known that TT binds specifically to gangliosides with a higher affinity to GDI b and GTIb (3). These sialo sphingolipids are especially abundant in nervous tissues. The question now arises: How is TT binding to gangliosides related to its neurotoxicity? We investigated the effect of the TT-ganglioside interaction in membranes by assaying the formation of transmembrane ionic channels in planar lipid bilayers. Asymmetric lipid bilayers (4) were formed by the apposition of a monolayer of asolectin (soybean phospholipids, AL) and a monolayer of AL supplemented with gangliosides (G). TT was added to one of the aqueous compartments and the current under voltage clamp was recorded. We found that TT forms channels only when added to the compartment in contact with the ganglioside face of the membrane. The TT channel is cation-selective and its residence time in the open state is longer than in the closed state. A preliminary account of this research appeared elsewhere (5). The interaction of TT with asymmetric lipid bilayers was investigated in two conditions (Fig. 1). The conductance of unmodified lipid bilayers was low (<4 pS). Addition of TT to the compartment limited by AL (lower panel) did not affect the membrane conductance even for periods longer than 1 h. In contrast, when TT was added to the compartment facing the gangliosides (upper panel), the membrane conductance increased in discrete steps and fluctuated between high and low conductance levels. This increase in conductance was due to the opening of ionic channels. Within minutes additional channels were inserted and the membrane conductance increased further. In addition, TT formed channels in symmetric bilayers containing gangliosides but not in symmetric bilayers composed exclusively of AL. Therefore, gangliosides are required for channel formation at neutral pH.'