MOLECULAR-STRUCTURE OF THE WATER CHANNEL THROUGH AQUAPORIN CHIP - THEHOURGLASS MODEL

Aquaporin channel-forming integral protein (CHIP) is the first charact erized water channel protein (genome symbol AQP1), but the molecular s tructure of the aqueous pathway through CHIP remains undefined. The tw o halves of CHIP are sequence-related, and each has three bilayer span ning domains with the motif asparagine-proline alanine (NPA) at residu es 76-78 (in cytoplasmic loop B) and 192-194 (in extracellular loop E) . The NPA motifs are oriented 180 degrees to each other, and the secon d NPA is near cysteine 189, the known site where mercurials inhibit os motic water permeability (P-f). When expressed in Xenopus oocytes, the double mutant A73C/C189S exhibited high, mercurial-sensitive P-f simi lar to wild-type CHIP. Conservative substitutions of slightly greater mass in or near NPA motifs in loop B or loop E in CHIP caused reduced P-f and failure of the protein to localize at the plasma membrane. Cer tain nonfunctional loop E mutants complemented the truncation mutant D 237Z. Formation of mixed oligomers was demonstrated by velocity sedime ntation, immunoprecipitation, and analysis of dimeric-CHIP polypeptide s. Cellular distributions of individual mutants or complementing pairs of mutants were verified by plasma membrane isolation and confocal mi croscopy. An hourglass structural model is proposed in which a cytopla smic chamber (loop B) connects within the membrane to an extracellular chamber (loop E) forming a single, narrow aqueous pathway through eac h of the CHIP subunits; subunit oligomerization may provide the vertic al symmetry necessary for residence within the lipid bilayer.