REACTION OF DIPHTHERIA-TOXIN CHANNELS WITH SULFHYDRYL-SPECIFIC REAGENTS - OBSERVATION OF CHEMICAL-REACTIONS AT THE SINGLE-MOLECULE LEVEL

The diphtheria toxin channel is believed to be a homooligomer of its T domain in which each subunit consists of two cy-helices, lying within the membrane, connected by a short interhelical loop of four amino ac ids (residues 349-352). To investigate the validity and implications o f this model, we singly mutated each of these amino acids to cysteines , formed channels with the mutant T-domain proteins in planar lipid bi layers, and added to the trans compartment sulfhydrylspecific reagents [methanethiosulfonate derivatives (MTS-ER)] that introduce a positive or negative charge to reacted cysteines. The introduction of a positi ve charge at residue 351 or 352 (through the MTS-ER reactions) resulte d in a step decrease in single-channel conductance, whereas the introd uction of a negative charge resulted in a step increase. The opposite sign of these effects indicates the predominantly electrostatic nature of the phenomenon and implies that residues 351 and 352 lie close to the channel entrance. The same reactions at residue 350 resulted in ve ry little change in channel conductance but instead changed the charac ter of the natural rapid flickering of the channel between open and cl osed states to one in which the channel spent more time in the closed state; this may have resulted from the group introduced at position 35 0 acting as a tethered channel blocker. The MTS derivatives had no eff ect on channels containing a cysteine at position 349, suggesting that this residue faces away from the channel entrance. We propose that th e step changes in conductance or flickering pattern result from the ch emical reaction of one MTS-ER molecule with one cysteine, and thus a b imolecular chemical reaction is being witnessed at the single molecule level. From the distribution of waiting times between the appearance (i.e., the opening) of a channel and the step change in its conductanc e or flickering pattern, we can calculate a pseudo-first-order rate co nstant, which can then be converted to a second-order rate constant, f or the chemical reaction.