Na+/H+ exchanger (NHE) is a major contributor in controlling intracellular pH. The activity of this protein is allosterically modified by intracellular H+. Histidine residues of the NHE that face the cytoplasm may be involved in determining the intracellular pH set point, with their state of protonation influencing the rate of Na +/H+ exchange. To test this hypothesis, histidine residues in the ubiquitously expressed NHE isoform (NHE1) that are relatively conserved amongst members of the NHE gene family were substituted by site-directed mutagenesis and the mutants were stably transfected into mammalian cells that are deficient in endogenous Na+/H + exchange activity. The pHi sensitivity of each mutant was evaluated by measuring the rate of 22Na + influx as a function of the intracellular H+ concentration. Mutation of the histidines located at the putative cytoplasmic face of the N-terminal transmembraneous domain of NHE1 did not show any significant effect on the pHi sensitivity of the protein. By contrast, substitution of histidines located in the C-terminal cytoplasmic tail activated the exchanger by increasing its sensitivity to H+. These mutants were no longer activated in response to protein kinase C, when compared to wild type. Taken together, these data support the hypothesis that some of the relatively conserved histidine residues in the C-terminal cytoplasmic tail of NHE1 may be involved in determining the pHi "threshold" or "set point" of the transporter.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.31524 |
| Date | January 2000 |
| Creators | Pazooki, Babak. |
| Contributors | Orlowski, John (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Physiology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001802394, proquestno: MQ70483, Theses scanned by UMI/ProQuest. |
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