title: Functional characterization and modified rescue of novel AE1 mutation R730C associated with overhydrated cation leak stomatocytosis.
abstract: We report the novel, heterozygous AE1 mutation R730C associated with dominant, overhydrated, cation leak stomatocytosis and well-compensated anemia. Parallel elevations of red blood cell cation leak and ouabain-sensitive Na(+) efflux (pump activity) were apparently unaccompanied by increased erythroid cation channel-like activity, and defined ouabain-insensitive Na(+) efflux pathways of nystatin-treated cells were reduced. Epitope-tagged AE1 R730C at the Xenopus laevis oocyte surface exhibited severely reduced Cl(-) transport insensitive to rescue by glycophorin A (GPA) coexpression or by methanethiosulfonate (MTS) treatment. AE1 mutant R730K preserved Cl(-) transport activity, but R730 substitution with I, E, or H inactivated Cl(-) transport. AE1 R730C expression substantially increased endogenous oocyte Na(+)-K(+)-ATPase-mediated (86)Rb(+) influx, but ouabain-insensitive flux was minimally increased and GPA-insensitive. The reduced AE1 R730C-mediated sulfate influx did not exhibit the wild-type pattern of stimulation by acidic extracellular pH (pH(o)) and, unexpectedly, was partially rescued by exposure to sodium 2-sulfonatoethyl methanethiosulfonate (MTSES) but not to 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET). AE1 R730E correspondingly exhibited acid pH(o)-stimulated sulfate uptake at rates exceeding those of wild-type AE1 and AE1 R730K, whereas mutants R730I and R730H were inactive and pH(o) insensitive. MTSES-treated oocytes expressing AE1 R730C and untreated oocytes expressing AE1 R730E also exhibited unprecedented stimulation of Cl(-) influx by acid pH(o). Thus recombinant cation-leak stomatocytosis mutant AE1 R730C exhibits severely reduced anion transport unaccompanied by increased Rb(+) and Li(+) influxes. Selective rescue of acid pH(o)-stimulated sulfate uptake and conferral of acid pH(o)-stimulated Cl(-) influx, by AE1 R730E and MTSES-treated R730C, define residue R730 as critical to selectivity and regulation of anion transport by AE1.
authors.affiliation: Division of Nephrology, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215, USA.
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Stewart
authors.forename: Prabhakar S
authors.initials: PS
authors.lastname: Kedar
authors.forename: Boris E
authors.initials: BE
authors.lastname: Shmukler
authors.forename: David H
authors.initials: DH
authors.lastname: Vandorpe
authors.forename: Ann
authors.initials: A
authors.lastname: Hsu
authors.forename: Bertil
authors.initials: B
authors.lastname: Glader
authors.forename: Alicia
authors.initials: A
authors.lastname: Rivera
authors.forename: Carlo
authors.initials: C
authors.lastname: Brugnara
authors.forename: Seth L
authors.initials: SL
authors.lastname: Alper
mesh_terms: D000328:Adult; D019943:Amino Acid Substitution; D000745:Anemia, Hemolytic, Congenital / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics*; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D005260:Female; D006021:Glycophorins / Q000096:biosynthesis; D006801:Humans; D015160:Hydrops Fetalis / Q000235:genetics / Q000378:metabolism; D007473:Ion Channels / Q000187:drug effects; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000235:genetics / Q000502:physiology; D008698:Mesylates / Q000494:pharmacology; D009154:Mutation; D009761:Nystatin / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D012413:Rubidium / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D004791:Enzyme Inhibitors; D006021:Glycophorins; D007473:Ion Channels; D007476:Ionophores; D008698:Mesylates; D013431:Sulfates; D009761:Nystatin; C074502:methanethiosulfonate; D010042:Ouabain; D012413:Rubidium