Mechanistic insight into pH gating in PIP aquaporins: role of specific loopD amino acids.

Abstract

Aquaporins are membrane channels that transport water and other solutes. Their transport activity is regulated by different stimuli such as pH, binding of cations, phosphorylation/dephosphorylation or interaction with other proteins. All aquaporins share a general fold and tetrameric quaternary structure. Each protomer present two conserved regions that regulate the specificity of transport: the Asn-Pro-Ala (NPA) motives and the aromatic/arginine (Ar/R) selectivity filter. The plant PIP subfamily of aquaporins distinguish from other members of the family due to a longer intracellular loopD which is involved in its gating mechanism. The open/closed conformational transition in PIP channels is triggered by intracellular acidification, and different pH0,5 are found for homotetramers and heterotertamers form by PIP1 and PIP2 paralogues. Our goal is to elucidate the role of specific LoopD’ s amino acids in the modulation of PIP gating mechanism by intracellular pH. Our in vitro and in silico experiments, for wild type and mutant PIP show that: i- a conserved Leu residue in the cytoplasmic constriction is the structural element that determines pore blockage and, ii- a Pro residue present in PIP2 but not in PIP1 channels is involved in differential pH0.5 of homo and heterotetramers dose-response curves. So, two loopD residues, Leu 206 and Pro 194, works in combination with the pH sensor His 202 to control effective pore closing.