Molecular basis of epithelial Cl channels

Proximal straight tubules (PST) were dissected from rabbit kidneys, held by crimping pipettes in a chamber and bathed in a buffered isosmotic (295 mOsm/kg) solution containing 200 mm mannitol (MBS). Changes in tubule diameter were monitored on line with an inverted microscope, TV camera and image processor. The PST were then challenged for 20 sec with MBS made 35 mOsm/kg hyperosmotic by addition of either NaCl, KCl, mannitol (M), glycerol (G), ethylene glycol (E), glycine (g), urea (U), acetamide (A) or formamide (F). With NaCl, KCl, M, G, E, g, U, and A, tubules shrunk osmometrically within 0.5 sec and remained shrunk for as long as 20 sec without recovering their original volume (sometimes A showed some recovery). PST barely shrunk with F and quickly recovered their original volume. The permeability coefficients were 0 m/sec (NaCl, M, g, E and U), 1 m/sec (A), 84 m/sec (F) and 0.02 m/sec (G). The reflection coefficients = 1.0 (NaCl, KCl, M, G, E, g and U), 0.95 (A) and 0.62 (F). Similar values were obtained by substituting 200 mOsm/kg M in MBS by either NaCl, KCl, G, E, g, U, a or F. The olive oil/water partition coefficients are 5 (M), 15 (U), 85 (A) and 75 (F) (all x10^–5). Thus, part of F permeates the cell membrane through the lipid bilayer. The probing molecules van der Waals diameters are 7.4×8.2×12.0 (M), 3.6×5.2×5.4 (U), 3.8×5.2 ×5.4 (A) and (3.4×4.5×5.4 (F) Å. We conclude that only F clearly permeates the water channel (WCH). Water molecules must single file within the WCH. After subtraction of the bilayer permeability of the probes, we estimate for the WCH selectivity filter cross-section a diameter of 4.2–4.7 Å (if it is circular) and 3.6×4.2 Å (if it is rectangular). But if the oxygens facing the WCH lumen H bond with the molecules crossing the WCH, the WCH selectivity filter would be 3.3–3.8 Å (circular) and 3.6×4.0 Å (rectangular).This work was supported in part from grants from CONICIT, Consejo de Desarrollo Científico y Humanístico of UCV and Fundación Polar.