Mercurial sensitivity of aquaporin 1 endofacial loop B residues

Citation
Ky. Kuang et al., Mercurial sensitivity of aquaporin 1 endofacial loop B residues, PROTEIN SCI, 10(8), 2001, pp. 1627-1634
Citations number
18
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN SCIENCE
ISSN journal
0961-8368 → ACNP
Volume
10
Issue
8
Year of publication
2001
Pages
1627 - 1634
Database
ISI
SICI code
0961-8368(200108)10:8<1627:MSOA1E>2.0.ZU;2-J
Abstract
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.