Nonradioactive monitoring of organic and inorganic solute transport into single Xenopus oocytes by capillary zone electrophoresis.
Transport of organic and inorganic solutes into and out of cells requires specialized transport proteins. Given a sufficiently sensitive analytical method for measuring cellular solute concentrations, it should be possible to monitor solute transport across the plasma membrane at the level of single cells. We report a capillary zone electrophoresis approach that is generally applicable to monitor solute transport into Xenopus laevis oocytes, requires only nanoliters of sample, and involves no radioactive materials. The sensitivity of capillary electrophoresis with UV detection is typically on the order of 10(-5)-10(-6) M, resulting in the mass detection limits in the low femtomole range. We show that capillary zone electrophoresis serves as a simple technique to measure solute transport into oocytes. Studies of the mammalian oligopeptide transporter PepT1 and the Na(+)- and K(+)-coupled epithelial and neuronal glutamate transporter EAAC1 expressed in oocytes demonstrate that transport of the dipeptide Trp-Gly via PepT1 and transport of Na+ and K+ via EAAC1 across the oocyte plasma membrane can be monitored by measuring intracellular tryptophan absorption and by indirect UV detection of inorganic ions, respectively. The CZE method allowed the simultaneous detection of changes of intracellular Na+ and K+ concentrations in response to EAAC1-mediated Na+ cotransport and K+ countertransport. This is the first report of a capillary zone electrophoresis-based quantitative analysis of intracellular components of a single cell in response to transport activity.
Nussberger
S
S
Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Foret
F
F
Hebert
S C
SC
Karger
B L
BL
Hediger
M A
MA
D027322:Amino Acid Transport System X-AG; D000818:Animals; D001693:Biological Transport, Active; D055592:Biophysical Phenomena; D001703:Biophysics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D050580:Excitatory Amino Acid Transporter 3; D005260:Female; D050491:Glutamate Plasma Membrane Transport Proteins; D006801:Humans; D066298:In Vitro Techniques; D017136:Ion Transport; D008845:Microinjections; D009865:Oocytes / Q000378:metabolism*; D000074011:Peptide Transporter 1; D011188:Potassium / Q000378:metabolism; D018075:RNA, Complementary / Q000008:administration & dosage / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D027981:Symporters; D014982:Xenopus laevis
D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D004151:Dipeptides; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D000074011:Peptide Transporter 1; D018075:RNA, Complementary; D011994:Recombinant Proteins; C082186:SLC15A1 protein, human; C493400:SLC1A1 protein, human; D027981:Symporters; D012964:Sodium; D011188:Potassium