title: Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S.
abstract: 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 (86)Rb(+), with secondarily increased (86)Rb(+) influx sensitive to ouabain and to bumetanide. Increased RhAG-associated (14)C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li(+), (86)Rb(+), and (14)C-MA were pharmacologically distinct, and Li(+) uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH(4)(+) and Gd(3+). RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH(3)/NH(4)(+), 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 NH(4)Cl, 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 NH(4)Cl or MA/MA(+). These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH(3)/NH(4)(+) 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 NH(3)/NH(4)(+)-associated depolarization; and 3) RhAG transports NH(3)/NH(4)(+) 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.
authors.affiliation: Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215, USA.
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Stewart
authors.forename: Boris E
authors.initials: BE
authors.lastname: Shmukler
authors.forename: David H
authors.initials: DH
authors.lastname: Vandorpe
authors.forename: Alicia
authors.initials: A
authors.lastname: Rivera
authors.forename: John F
authors.initials: JF
authors.lastname: Heneghan
authors.forename: Xiaojin
authors.initials: X
authors.lastname: Li
authors.forename: Ann
authors.initials: A
authors.lastname: Hsu
authors.forename: Margaret
authors.initials: M
authors.lastname: Karpatkin
authors.forename: Allison F
authors.initials: AF
authors.lastname: O'Neill
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Bauer
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Heeney
authors.forename: Kathryn
authors.initials: K
authors.lastname: John
authors.forename: Frans A
authors.initials: FA
authors.lastname: Kuypers
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Gallagher
authors.forename: Samuel E
authors.initials: SE
authors.lastname: Lux
authors.forename: Carlo
authors.initials: C
authors.lastname: Brugnara
authors.forename: Connie M
authors.initials: CM
authors.lastname: Westhoff
authors.forename: Seth L
authors.initials: SL
authors.lastname: Alper
mesh_terms: D001483:Base Sequence; D001798:Blood Proteins / Q000235:genetics*; D015153:Blotting, Western; D002648:Child; D004912:Erythrocytes / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006801:Humans; D006947:Hyperkalemia / Q000151:congenital* / Q000235:genetics / Q000503:physiopathology; D007223:Infant; D007231:Infant, Newborn; D017136:Ion Transport / Q000235:genetics*; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics*; D008744:Methylamines / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D018408:Patch-Clamp Techniques; D010641:Phenotype
chemical_list: D001798:Blood Proteins; D008562:Membrane Glycoproteins; D008744:Methylamines; C498523:RHAG protein, human; C027451:methylamine