Water and ion permeation of aquaporin-1 in planar lipid bilayers. Major differences in structural determinants and stoichiometry.
American Society for Biochemistry and Molecular Biology
The aquaporin-1 (AQP1) water channel protein is known to facilitate the rapid movement of water across cell membranes, but a proposed secondary role as an ion channel is still unsettled. Here we describe a method to simultaneously measure water permeability and ion conductance of purified human AQP1 after reconstitution into planar lipid bilayers. Water permeability was determined by measuring Na(+) concentrations adjacent to the membrane. Comparisons with the known single channel water permeability of AQP1 indicate that the planar lipid bilayers contain from 10(6) to 10(7) water channels. Addition of cGMP induced ion conductance in planar bilayers containing AQP1, whereas cAMP was without effect. The number of water channels exceeded the number of active ion channels by approximately 1 million-fold, yet p-chloromethylbenzenesulfonate inhibited the water permeability but not ion conductance. Identical ion channel parameters were achieved with AQP1 purified from human red blood cells or AQP1 heterologously expressed in Saccharomyces cerevisae and affinity purified with either N- or C-terminal poly-histidine tags. Rp-8-Br-cGMP inhibited all of the observed conductance levels of the cation selective channel (2, 6, and 10 pS in 100 mm Na(+) or K(+)). Deletion of the putative cGMP binding motif at the C terminus by introduction of a stop codon at position 237 yielded a truncated AQP1 protein that was still permeated by water but not by ions. Our studies demonstrate a method for simultaneously measuring water permeability and ion conductance of AQP1 reconstituted into planar lipid bilayers. The ion conductance occurs (i) through a pathway distinct from the aqueous pathway, (ii) when stimulated directly by cGMP, and (iii) in only an exceedingly small fraction of AQP1 molecules.
Lipid Bilayers, Cell Membrane Permeability, Aquaporins/metabolism
J Biol Chem 2001 Aug 24;276(34):31515-20. Epub 2001 Jun 15. doi: 10.1074/jbc.M104267200