The water channel protein aquaporin-1 (AQP1) has two asparagine-proline-ala
nine (NPA) repeats on loops B and E. From recent structural information, th
ese loops are on opposite sides of the membrane and meet to form a pore. We
replaced the mercury-sensitive residue cysteine 189 in AQP1 by serine to o
btain a mercury-insensitive template (C189S). Subsequently, we substituted
three consecutive cysteines for residues 71-73 near the first NPA repeat (7
6-78) in intracellular loop B, and investigated whether they were accessibl
e to extracellular mercurials. AQP1 and its mutants were expressed in Xenop
us laevis oocytes, and the osmotic permeability (P-f) of the oocytes was de
termined. C189S had wild-type P-f but was not sensitive to HgCl2. Expressio
n of all three C189S cysteine mutants resulted in increased P-f, and all th
ree mutants regained mercurial sensitivity. These results, especially the i
nhibitions by the large mercurial p-chloromer-cunbenzene-sulfonic acid (pCM
BS) (similar to6 Angstrom wide), suggest that residues 71-73 at the pore ar
e accessible to extracellular mercurials. A 30-ps molecular dynamics simula
tion (at 300 K) starting with crystallographic coordinates of AQP1 showed t
hat the width of the pore bottleneck (between Connolly surfaces) can vary (
w(avg) = 3.9 Angstrom, sigma = 0.75; hydrated AQP1). Thus, although the por
e width would be greater than or equal to 6 Angstrom only for 0.0026 of the
time, this might suffice for pCMBS to reach residues 71-73. Alternative ex
planations such as passage of pCMBS across the AQP1 tetramer center or othe
r unspecified transmembrane pathways cannot be excluded.