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Physiology of ion-transport
processes involved in the regulation of intracellular pH. Our
work focuses on the ion-transport processes involved in regulation of intracellular
pH (pHi), and how these transporters are themselves regulated by cell volume,
hormones, and oncogenes. Our laboratory uses pH-sensitive microelectrodes and
dyes (including digital imaging techniques) to monitor pHi in single cells (e.g.,
neurons, glia, muscle, mesangial cells, osteoclasts, and cells of perfused renal
tubules, gastric glands and colonic crypts). The primary objective is to deduce
mechanisms by which acids and bases are transported across membranes, how the
transporters are regulated, and how pHi changes affect processes such as growth
control, transepithelial acid-base transport, and the tone of vascular smooth
muscle. We are also expressing the electrogenic acid-base transporters that are
responsible for glutamate uptake in the nervous system, as well as oligopeptide
uptake. Finally, we are attempting to clone the genes for bicarbonate transport
proteins. Figure caption: Effect
of long-term expression of the oncogene c-H-ras on transporters that alkalinize
cells.
Measuring intracellular pH (pHi) with a fluorescent dye, we found that
fibroblasts transfected with ras have a pHi of ~7.5, half a pH unit higher than
untransfected parental cells. (A) ras speeds the pHi recovery from an intracellular
acid load imposed by applying and then withdrawing a solution containing NH3 and
NH4+. At each pHi, the pHi recovery rate is faster in the ras-transfected cells
(ab vs a'b'). (B) In ras-transfected cells (filled symbols), the two transporters
responsible for the pHi recovery in panel A are greatly stimulated. Removing specific
ions or applying inhibitors, we determined how the Na-H exchange and the Na+-dependent
Cl-HCO3 exchange rates (fluxes) vary with pHi. ras transfection causes resting
pHi to be abnormally high, and the pHi recovery rate to be extremely fast, because
ras shifts the flux-pHi profile of both transporters ~0.7 pH units in the alkaline
direction (open vs filled symbols). At any pHi, the transporters are far more
active in ras cells, perhaps explaining why some cancer cells are able to survive
in the acidic environment of a tumor.
Selected publications:
Toye AM, Parker MD, Daly CM, Lu J, Virkki LV, Pelletier MF, Boron WF. The human NBCe1-A mutant R881C, associated with proximal renal tubular acidosis, retains function but is mistargeted in polarized renal epithelia. AM J Physiol Cell Physiol. 2006 May 17, ahead of print.
Zhou Y, Bouyer P, Boron WF. Role of a tryrosine kinase in the CO2-induced stimulation of HCO-3 reabosroption by rabbit S2 proximal tubules. Am J Physiol Renal Physiol. 2006 May 16, head of print.
Lu J, Daly CM, Parker MD, Gill HS, Piermarini PM, Pelletier MF, Boron WF. Effect of human carbonic anhydrase II on the activity of the human electrogenic Na/HCO3 cotransporter NBC10A in Xenopus oocytes. J Biol Chem, 2006 May 10, ahead of print.
Romero MF, Fulton CM, Boron WF. The SLC4 family of HCO3-transporters. Pflugers Arch. 2004 447:495-509.
Grichtchenko
II, Choi I, Zhong X, Bray-Ward P, Russell JM, Boron WF. Nucleotide
Cloning, characterization, and chromosomal mapping of a human electroneutral Na+-driven
Cl-HCO3 exchanger. J Biol Chem. 2001 Mar 16;276(11):8358-63
Maunsbach
AB, Vorum H, Kwon TH, Nielsen S, Simonsen B, Choi I, Schmitt BM, Boron WF, Aalkjaer
C. Immunoelectron
microscopic localization of the electrogenic Na/HCO3 cotransporter in rat and
ambystoma kidney. J Am Soc Nephrol. 2000 Dec;11(12):2179-89.
Schmitt
BM, Berger UV, Douglas RM, Bevensee MO, Hediger MA, Haddad GG, Boron WF. Na/HCO3
cotransporters in rat brain: expression in glia, neurons, and choroid plexus. J Neurosci. 2000 Sep 15;20(18):6839-48.
walter.boron@yale.edu
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