We examined the hypothesis that Na+/H+ exchanger expression is regulated during fetal and neonatal development and differentiation. To examine transcriptional regulation of the NHE1 isoform of the Na+/H+ exchanger, transgenic mice were created that contained the mouse NHE1 promoter driving expression of green fluorescent protein. The level of NHE1 transcription varied between tissues and with the stage of embryonic development. The highest expression was in the heart and liver of 12- to 15-day-old mice, and this declined with age. To examine Na+/H+exchanger protein levels, we immunoblotted mouse tissues from 18-day-old embryos, neonates, and adults. Protein levels increased after embryonic day 18 and peaked at 14 days of age in the heart, lung, liver, kidney, and brain. The greatest rise in NHE1 protein expression occurred in the heart, whereas the smallest increase was in the brain. The results suggest that Na+/H+ exchanger transcription and protein levels are controlled in a tissue-specific and time-dependent manner during development.

In this paper we examine the role of thyroid hormone in regulating expression of the Na ؉ /H ؉ exchanger. Thyroid hormone has been reported to regulate the activity of the Na ؉ /H ؉ exchanger messenger RNA in some cell types. Treatment of cardiac myocytes with 3,5,3-triiodothyronine results in an increased expression of Na ؉ /H ؉ exchanger protein. Also, compared with euthyroid animals, hypothyroid rats express decreased amounts of the Na ؉ /H ؉ exchanger protein. To examine the mechanisms involved in regulating expression of the Na ؉ /H ؉ exchanger, we have characterized the regulation of a distal element of the NHE1 promoter by the thyroid hormone receptor. We have previously shown that a ؊1085/؊800 nucleotide (nt) region of the promoter is a modular element with a ؊841/؊800 nt activating element. Using electrophoretic mobility shift assay, we show that this element interacts with thyroid hormone receptor TR␣ 1 , a nuclear hormone receptor. The addition of exogenous TR␣ increased transcriptional activity of the ؊841/؊800 nt element of the Na ؉ /H ؉ exchanger promoter. We show that TR␣ binds to a region on the ؊841/؊800 nt element that is near, but not identical, to the previously identified chicken ovalbumin upstream promoter transcription factor-binding site. Our results are the first demonstration that thyroid hormone and the thyroid hormone receptor TR␣ 1 regulate expression of the Na ؉ /H ؉ exchanger.

No abstract

The Na+/H+ exchanger is an integral membrane protein found in the plasma membrane of eukaryotic and prokaryotic cells. In eukaryotes it functions to exchange one proton for a sodium ion. In mammals it removes intracellular protons while in plants and fungal cells the plasma membrane form removes intracellular sodium in exchange for extracellular protons. In this study we used the Na+/H+ exchanger of Schizosaccharomyces pombe (Sod2) as a model system to study amino acids critical for activity of the protein. Twelve mutant forms of the Na+/H+ exchanger were examined for their ability to translocate protons as assessed by a Cytosensor microphysiometer. Mutation of the amino acid Histidine 367 resulted in defective proton translocation. The acidic residues Asp145, Asp178, Asp266 and Asp267 were important in the proton translocation activity of the Na+/H+ exchanger. Mutation of amino acids His98, His233 and Asp241 did not significantly impair proton translocation by the Na+/H+ exchanger. These results confirm that polar amino acids are important in proton flux activity of Na+/H+ exchangers.