Water and ion permeation of aquaporin-1 in planar lipid bilayers - Major differences in structural determinants and stoichiometry

The aquaporin-1 (AQP1) water channel protein is known to facilitate the rap id movement of water across cell membranes, but a proposed secondary role a s an ion channel is still unsettled. Here we describe a method to simultane ously measure water permeability and ion conductance of purified human AQP1 after reconstitution into planar lipid bilayers. Water permeability was de termined by measuring Na+ concentrations adjacent to the membrane. Comparis ons with the known single channel water permeability of AQP1 indicate that the planar lipid bilayers contain from 10(6) to 10(7) water channels. Addit ion of cGMP induced ion conductance in planar bilayers containing AQP1, whe reas cAMP was without effect. The number of water channels exceeded the num ber of active ion channels by approximately 1 million-fold, yet p-chloromet hylbenzenesulfonate inhibited the water permeability but not ion conductanc e. Identical ion channel parameters were achieved with AQP1 purified from h uman red blood cells or AQP1 heterologously expressed in Saccharomyces cere visae and affinity purified with either N- or C-terminal poly-histidine tag s. Rp-8-Br-cGMP inhibited all of the observed conductance levels of the cat ion selective channel (2, 6, and 10 pS in 100 mM Na+ or K+). Deletion of th e putative cGMP binding motif at the C terminus by introduction of a stop c odon at position 237 yielded a truncated AQP1 protein that was still permea ted by water but not by ions. Our studies demonstrate a method for simultan eously measuring water permeability and ion conductance of AQP1 reconstitut ed into planar lipid bilayers. The ion conductance occurs (i) through a pat hway distinct from the aqueous pathway, (ii) when stimulated directly by cG MP, and (iii) in only an exceedingly small fraction of AQP1 molecules.