Highly selective water channel activity measured by voltage clamp: Analysis of planar lipid bilayers reconstituted with purified AqpZ

Aquaporins are membrane channels selectively permeated by water or water pl us glycerol. Conflicting reports have described ion conductance associated with some water channels, raising the question of whether ion conductance i s a general property of the aquaporin family. To clarify this question, a d efined system was developed to simultaneously measure water permeability an d ion conductance. The Escherichia coli water channel aquaporin-Z (AqpZ) wa s studied, because it is a highly stable tetramer. Planar lipid bilayers we re formed from unilamellar vesicles containing purified AqpZ. The hydraulic conductivity of bilayers made from the total extract of E. coli lipids inc reased 3-fold if reconstituted with AqpZ, but electric conductance was unch anged. No channel activity was detected under voltage-clamp conditions, ind icating that less than one in 10(9) transport events is electrogenic. Micro electrocle measurements were simultaneously undertaken adjacent to the memb rane. Changes in sodium concentration profiles accompanying transmembrane w ater flow permitted calculation of the activation energies: 14 kcal/mol for protein-free lipid bilayers and 4 kcal/mol for lipid bilayers containing A qpZ. Neither the water permeability nor the electric conductivity exhibited voltage dependence. This sensitive system demonstrated that AqpZ is permea ted by water but not charged ions and should permit direct analyses of puta tive electrogenic properties of other aquaporins.