The mobility of single-file water molecules is governed by the number of H-bonds they may form with channel-lining residues

Abstract

Channel geometry governs the unitary osmotic water channel permeability, p_f, according to classical hydrodynamics. Yet, p_f varies by several orders of magnitude for membrane channels with a constriction zone that is one water molecule in width and four to eight molecules in length. We show that both the p_f of those channels and the diffusion coefficient of the single-file waters within them are determined by the number N_H of residues in the channel wall that may form a hydrogen bond with the single-file waters. The logarithmic dependence of water diffusivity on N_H is in line with the multiplicity of binding options at higher N_H densities. We obtained high-precision p_f values by (i) having measured the abundance of the reconstituted aquaporins in the vesicular membrane via fluorescence correlation spectroscopy and via high-speed atomic force microscopy, and (ii) having acquired the vesicular water efflux from scattered light intensities via our new adaptation of the Rayleigh-Gans-Debye equation.

Fig. 1. Determination of reconstitution efficiency.

(A) FCS autocorrelation curves allowed us to obtain the number of (i) vesicles labeled with 0.004% (w/w) N-(lissamine-rhodamine-sulfonyl)phosphatidylethanolamine (sandy brown), (ii) AQP1-YFP–containing vesicles (purple), (iii) AQP1-YFP oligomers containing micelles that formed upon vesicle dissolution in mild detergent (dashed purple), and (iv) AQP1-YFP monomer containing micelles that formed upon further dissolution in harsh detergent (dotted purple) per confocal volume. The buffer (pH 7.4) contained 100 mM NaCl, 20 mM Mops, and a protease inhibitor. (B and C) AFM imaging of solid-supported lipid bilayers that were prepared from AQPZ proteoliposomes (B) or empty vesicles (investigated area: 14 × 400 × 400nm²) (C) resulted in histograms of height values (n = 50). (B) Inset: The high-resolution raw data allowed differentiation of the extracellular and cytoplasmic AQPZ surfaces. (C) The density of unspecified features (0.218 per vesicle) served to correct the protein count (see also fig. S1). (D) Comparison of both the absolute AFM and FCS counts of AQPZ tetramers per liposome (at three different concentrations) (upper panel) and their ratio (middle panel). Average ratio of AFM and FCS counts per liposome for AQPZ, GlpF, and AQP1 oligomers (lower panel: compare also eqs. S9 and S10).

Fig. 2. The osmotic shrinkage of proteoliposomes.

(A) Representative stopped-flow raw data (spline lines) for AQPZ and the fit (dashed lines) according to Eq. 4. Equal volumes of vesicle suspension and hyperosmotic solution (300 mM sucrose) were mixed (5°C, same buffer as in Fig. 1). The number of reconstituted AQP monomers per proteoliposome is indicated. (B) P_f of reconstituted vesicles was calculated as shown in (A) for at least three independently purified and reconstituted batches for each protein and plotted as a function of the channel number per proteoliposome.

Fig. 3. Water movement in aquaporins.

(A) p_f (at 5°C) for AQPZ, GlpF, and AQP1 was taken from the slopes in Fig. 2B. The background water permeability of a single lipid vesicle was calculated by multiplying P_f,l by the surface area of the vesicle. (B) The diffusion coefficient D_W (25°C) of water molecules inside the channel was calculated (Eq. 1). The bulk water diffusion coefficient is shown for comparison.

Fig. 4. Number N_H of pore-lining residues (in yellow circles) that may form hydrogen bonds (dotted lines) with single-file water molecules.

The 0.88-Å resolution structure of yeast AQP1 (33) [Protein Data Bank (PDB) #3Z0J] served as a template to find a model for the AQP1 (PDB #1J4N), AQPZ (PDB #1RC2), and GlpF (PDB #1FX8) structures via the PyMol’s “align” routine (34). The position of the water molecules (red spheres) is from the yeast AQP1 structure. The two water molecules below R206 have been added in the GlpF model to indicate that this region is wide enough to let the water molecules bypass each other within the pore.

Fig. 5. D_W depends on the number N_H of hydrogen bonds that single-file water molecules may form with pore-lining residues.

D_W in nanotubes assumes z = 2.6 Å and k₀ = 0.1 p s⁻¹ (13). p_f for the bacterial potassium channel KcsA was calculated by applying Eq. 4 to our previously published stopped-flow curves (18). Equation 1 served to compute D_W from the p_f values of gramicidin, midigramicidin, minigramicidin (17), and KcsA.