Length of the selectivity filter of aquaporin‐1*

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Abstract
We have characterized the selectivity filter of the water channel aquaporin‐1 (AQP1) of proximal straight tubules (PST), as an equivalent cylindrical structure with a diameter of ∼ 4.5 Å, where water molecules single file. We report here efforts to evaluate its length. PST were dissected from rabbit kidneys, held with pipettes in a chamber bathed in a buffered mannitol isosmotic solution (MBS, 295 mOsm/kg). Changes in tubule cell volume with time (dV/Adt), were monitored, on line, with an inverted microscope, a TV camera and an image processor. Osmotic permeability coefficients, Pos, and reflection coefficients (σs) were measured with several solutes: mannitol (M), raffinose (R), sucrose (S), glycerol (G), acetamide (A) and urea (U). For this purpose PST were suddenly exposed (in ∼ 80 ms and for 20 s) to a hyperosmolality step (ΔCs) achieved by adding to MBS a ΔCs of 35 mOsm/kg of either R, S, M, G, A or U. Cells shrunk within 500 ms of t = 0 to their osmometric volume and remained shrunk for the 20 s of the ΔCs. Pos was measured from the shrinking curves; Pos = 3000 ± 25 μm/s with either R, S, M, G, A or U. This procedure also allowed to calculate σs; σs = 1.00 for R, S, M, G, A and U, indicating that these solutes do not penetrate the water channel. In contrast, the shrinking curves produced by a ΔCs = 35 mOsm/kg formamide (F) were 15th to 16th slower and smaller (subosmometric) than those produced by a ΔCs = 35 mOsm/kg of R, S, M, G, A or U. Furthermore, with F, cells did not remain shrunk. They recovered their original volume within 3 s. Pos (measured with F) is denoted as Pos*; Pos* = 480 ± 30 μm/s. σs,formamide (denoted σsp) = 0.16 ± 0.01. Use of σsp and Pos* values in Hill's equations for the bimodal theory of osmosis leads to n = 2–3, n being the number of water molecules single filing within the channel selectivity filter, whose length must lie within 6 to 9 Å, a value lower than previous values calculated from the Pos Pd* ratio.

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