Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S

Stewart AK, Shmukler BE, Vandorpe DH, Rivera A, Heneghan JF, Li X, Hsu A, Karpatkin M, O'Neill AF, Bauer DE, Heeney MM, John K, Kuypers FA, Gallagher PG, Lux SE, Brugnara C, Westhoff CM, Alper SL. Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S. Am J Physiol Cell Physiol 301: C1325-C1343, 2011. First published August 17, 2011; doi:10.1152/ajpcell.00054.2011.-Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wild-type RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li+ and Rb-86(+), with secondarily increased Rb-86(+) influx sensitive to ouabain and to bumetanide. Increased RhAG-associated C-14-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li+, Rb-86(+), and C-14-MA were pharmacologically distinct, and Li+ up-takes associated with RhAG and RhAG F65S were differentially inhibited by NH4+ and Gd-3(+). RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH3/NH4+, but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA(+)). RhAG F65S-expressing oocytes exhibited near-wild-type responses to NH4Cl, but MA/MA(+) elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li+ substitution or bath addition of 5 mM NH4Cl or MA/MA(+). These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH3/NH4+ and MA/MA(+); 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA(+) transport, and decreased NH3/NH4+ -associated depolarization; and 3) RhAG transports NH3/NH4+ and MA/MA(+) by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte intracellular pH, membrane potential, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms.