Lumen geometry of ion channels formed by Vibrio cholerae EL Tor cytolysin elucidated by nonelectrolyte exclusion

Vibrio cholerae EL Tor cytolysin, a water-soluble protein with a molecular mass of 63 kDa, forms small pores in target cell membranes, In this communi cation, planar lipid bilayers under voltage clamp conditions were used to i nvestigate the geometric properties of the pores. It was established that a ll cytolysin channels were inserted into membranes with the same orientatio n. Sharp asymmetry in the I-V curve of fully open cytolysin channels persis ting at high electrolyte concentrations indicated asymmetry in the geometry of the channel lumen. Using the nonelectrolyte exclusion method, evidence was obtained that the cis opening of the channel had a larger diameter (les s than or equal to1.9 nm) than the trans opening (less than or equal to1.6 nm). The channel lumen appeared constricted, with a diameter of less than o r equal to 1.2 nm. Cup-shaped lumen geometry was deduced for both channel o penings, which appeared to be connected to each other via a central narrow part. The latter contributed significantly to the total electrical resistan ce and determined the discontinuous character of channel filling with nonel ectrolytes. Comparisons of the properties of pores formed by cytolysins of two V. cholerae biotypes (EL Tor and non-Ol) indicated that the two ion cha nnels possessed a similar geometry. (C) 2001 Elsevier Science B.V. All righ ts reserved.