Aquaporin-4 (AQP4), the primary water channel in glial cells of the mammalian brain, plays a critical role in water transport in the central nervous system. Previous experiments have shown that the water permeability of AQP4 depends on the cholesterol content in the lipid bilayer, but it was not clear whether changes in permeability were due to direct cholesterol-AQP4 interactions or to indirect effects caused by cholesterol-induced changes in bilayer elasticity or bilayer thickness. To determine the effects resulting only from bilayer thickness, here we use a combination of experiments and simulations to analyze AQP4 in cholesterol-free phospholipid bilayers with similar elastic properties but different hydrocarbon core thicknesses previously determined by x-ray diffraction. The channel (unit) water permeabilities of AQP4 measured by osmotic-gradient experiments were 3.5 ± 0.2 × 10−13 cm3/s (mean ± SE), 3.0 ± 0.3 × 10−13 cm3/s, 2.5 ± 0.2 × 10−13 cm3/s, and 0.9 ± 0.1 × 10−13 cm3/s in bilayers containing (C22:1)(C22:1)PC, (C20:1)(C20:1)PC, (C16:0)(C18:1)PC, and (C13:0)(C13:0)PC, respectively. Channel permeabilities obtained by molecular dynamics (MD) simulations were 3.3 ± 0.1 × 10−13 cm3/s and 2.5 ± 0.1 × 10−13 cm3/s in (C22:1)(C22:1)PC and (C14:0)(C14:0)PC bilayers, respectively. Both the osmotic-gradient and MD-simulation results indicated that AQP4 channel permeability decreased with decreasing bilayer hydrocarbon thickness. The MD simulations also suggested structural modifications in AQP4 in response to changes in bilayer thickness. Although the simulations showed no appreciable changes to the radius of the pore located in the hydrocarbon region of the bilayers, the simulations indicated that there were changes in both pore length and α-helix organization near the cytoplasmic vestibule of the channel. These structural changes, caused by mismatch between the hydrophobic length of AQP4 and the bilayer hydrocarbon thickness, could explain the observed differences in water permeability with changes in bilayer thickness.
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