Properties of the Na+/H+ exchanger protein. Detergent-resistant aggregation and membrane microdistribution.
The Na+/H+ exchanger is a ubiquitous membrane protein of bacteria, plants and mammals. The first isoform discovered (NHE1) is present on the mammalian plasma membrane and transports one H+ out of cells in exchange for one extracellular Na+. With solubilization in standard SDS/PAGE buffer, this protein had a high tendency to aggregate when subjected to elevated temperature. The aggregates were stable and did not dissociate in high concentrations of SDS or 2-mercaptoethanol. We examined the distribution of the Na+/H+ exchanger within membrane subfractions. The Na+/H+ exchanger was found both in caveolin-containing fractions and, in lesser amounts, in higher density membrane fractions where the bulk of proteins were contained. Treatment with cytochalasin D caused only a minor reduction of the amount of Na+/H+ exchanger present in caveolin-enriched fractions suggesting an intact cytoskeleton was not important for NHE1 localization to these microdomains. Treatment of cells with methyl beta-cyclodextrin had a small stimulatory effect on Na+/H+ exchanger activity and reduced the amount of Na+/H+ exchanger in low density membrane fractions. Our study demonstrates that SDS cannot maintain the protein in a monomeric state suggesting that strong hydrophobic interactions are responsible for this temperature dependent aggregation behavior. In addition a large proportion of the Na+/H+ exchanger protein is found to be enriched in low density caveolin-containing fractions.
D000818:Animals; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents; D066298:In Vitro Techniques; D051379:Mice; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution; D014162:Transfection
C495541:Cav1 protein, mouse; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D003902:Detergents; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D012967:Sodium Dodecyl Sulfate