Human RhAG ammonia channel is impaired by the Phe65Ser mutation in overhydrated stomatocytic red cells

Genetet S, Ripoche P, Picot J, Bigot S, Delaunay J, Armari-Alla C, Colin Y, Mouro-Chanteloup I. Human RhAG ammonia channel is impaired by the Phe65Ser mutation in overhydrated stomatocytic red cells. Am J Physiol Cell Physiol 302: C419-C428, 2012. First published October 19, 2011; doi:10.1152/ajpcell.00092.2011.-In red cells, Rh-associated glycoprotein (RhAG) acts as an ammonia channel, as demonstrated by stopped-flow analysis of ghost intracellular pH (pH(i)) changes. Recently, overhydrated hereditary stomatocytosis (OHSt), a rare dominantly inherited hemolytic anemia, was found to be associated with a mutation (Phe65Ser or Ile61Arg) in RHAG. Ghosts from the erythrocytes of four of the OHSt patients with a Phe65Ser mutation were resealed with a pH-sensitive probe and submitted to ammonium gradients. Alkalinization rate constants, reflecting NH3 transport through the channel and NH3 diffusion unmediated by RhAG, were deduced from time courses of fluorescence changes. After subtraction of the constant value found for Rh-null lacking RhAG, we observed that alkalinization rate constant values decreased similar to 50% in OHSt compared with those of controls. Similar RhAG expression levels were found in control and OHSt. Since half of the expressed RhAG in OHSt most probably corresponds to the mutated form of RhAG, as expected from the OHSt heterozygous status, this dramatic decrease can be therefore related to the loss of function of the Phe65Ser-mutated RhAG monomer.