TY - JOUR T1 - The Activity of Human Aquaporin 1 as a cGMP-gated Cation Channel is Regulated by Tyrosine Phosphorylation in the Carboxyl Terminal Domain JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.111.073692 SP - mol.111.073692 AU - Ewan M Campbell AU - Dawn N Birdsell AU - Andrea J Yool Y1 - 2011/01/01 UR - http://molpharm.aspetjournals.org/content/early/2011/10/17/mol.111.073692.abstract N2 - In addition to a constitutive water channel activity, several studies suggest Aquaporin-1 (AQP1) functions as a non-selective monovalent cation channel activated by intracellular cGMP, though variability in responsiveness between preparations has led to controversy in the field. Data here support the hypothesis that responsiveness of the AQP1 ionic conductance to cGMP is governed by tyrosine phosphorylation. Wild type and mutant human AQP1 channels expressed in Xenopus oocytes were characterized by two-electrode voltage clamp and optical osmotic swelling analyses. Quadruple mutation by site-directed mutagenesis of barrier hydrophobic residues (V50, L54, L170, L174) to alanines in the central pore induced inward rectification of the ionic current and shifted reversal potential approximately +10mV, indicating increased permeability of tetraethylammonium ion. Introduction of cysteine at lysine 51 in the central pore (K51C) in a cysteine-less template created new sensitivity to block of the conductance by mercuric ion. Mutations of candidate consensus sites and pharmacological manipulation of serine and threonine phosphorylation did not alter cGMP-dependent responses; however, mutation of tyrosine Y253C or pharmacological dephosphorylation prevented ion channel activation. Modification of Y253C by covalent addition of a negatively charged group (MTSES) rescued the cGMP-activated conductance response, an effect reversed by dithiothreitol. Results support the proposal that phosphorylation of tyrosine Y253 in the carboxyl terminal domain, confirmed by western blot, acts as a master switch regulating responsiveness of AQP1 ion channels to cGMP, and the tetrameric central pore is the ion permeation pathway. These findings advance resolution of a standing controversy and expand our understanding of AQP1 as a multifunctional regulated channel. ER -