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Physiological processes such as acid-base
homeostasis, cell volume regulation, fluid secretion, and salt and water
absorption across epithelia are mediated in part by a family of
electroneutral membrane transporters that exchange extracellular sodium
(Na+) for cytoplasmic protons (H+) (Na+/H+ exchangers or NHEs).
Additionally, these transporters have been implicated in events underlying
cellular adhesion, migration, proliferation, transformation and apoptosis.
Dysfunction of certain NHE isoforms is associated with the pathogenesis of
several disease states, including essential hypertension, congenital
secretory diarrhea, diabetes, as well as tissue (notably cardiac and
neuronal) injuries resulting from episodes of ischemia/reperfusion.rom
episodes of ischemia/reperfusion.
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NHE molecule |
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Evolutionary diversity of sodium proton
exchangers |
The focus of
our research over the last decade has been to investigate the biological
basis for this functional heterogeneity in order to better understand
the role of NHEs in human health and disease. To this end,
low-stringency-hybridization cDNA cloning and cell transfection
approaches were initially used to isolate and characterize several NHE
genes from rat (RnNHE1-4) and human (HsNHE5). With the advent of the
human genome project, additional novel NHE-related genes (HsNHE6-9) have
been recently cloned and partially characterized.
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Their deduced primary structures are quite divergent (~ 25-70% amino
acid identity) and show considerable differences in their patterns of
tissue expression. To date, our studies using heterologous mammalian
expression systems have revealed striking differences in their
subcellular localization, kinetic properties, sensitivity to
pharmacological antagonists, and responsiveness to various signaling
pathways. In general, these isoforms can be subdivided into two classes:
those that are localized primarily to discrete regions of the plasma
membrane (e.g., apical or basolateral membranes of epithelial cells;
axons or dendrites of neuron; intercalated disks and t-tubules of
cardiac myocytes), and those that localize predominantly to distinct
intracellular organelles (e.g.; trans-Golgi network, endosomal
vesicles).
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NHE1 in cardiac myocytes
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Accumulation of NHE7 (red) in the trans-Golgi network and
associated vesicles. Mitochondria are stained with green fluorescent
protein (GFP). |
Over
the next several years, we intend to utilize a multifaceted approach –
incorporating a range of molecular, cellular and physiological techniques –
to address several distinct aspects of NHE biology, some of which are listed
below.
(1)
To define the transmembrane organization and structural domains of the
exchanger responsible for drug recognition and cation translocation in order
to develop a molecular model that accounts for the functional dynamics of
this transporter. The NHE1 isoform is being used as a model system since it
resides exclusively in the plasma membrane and expresses at high levels in
transfected CHO cells. This is being accomplished by site-directed and
cysteine-scanning mutagenesis of the NHE1 cDNA, stable gene expression in
mammalian cell lines, and by measuring of its transport properties using
established cation flux assays.
(2)
To investigate the physiological role and importance of the NHE1 isoform in
cardiac pH homeostasis which is critically important for myocardial
contractility, and to examine its involvement in injuries that result from
episodes of ischemia and reperfusion. Isolated intact hearts and cultured
cardiac myocytes are being utilized in these studies.
(3)
To define the protein sorting and signalling mechanisms that underlie
membrane targeting and regulation, respectively, of the different NHEs
(e.g., NHE1 to intercalated disks; NHE3 to the apical membrane and recycling
endosomes of epithelia; NHE5 to the plasma membrane and synaptic-like
vesicles of neurons; NHE6-9 to other discrete endomembrane compartments).
This is being accomplished by immunofluorescence confocal microscopy and
subcellular fractionation analyses. The elements responsible for membrane
targeting and regulation are being resolved by construction of chimeric
molecules, and by deletion and site-directed mutagenesis. Interacting
proteins are being identified by yeast two-hybrid and proteomic approaches.
(
4) To
evaluate the physiological roles of novel organellar NHEs to cellular
function. The importance of NHE6-9 to organellar function and morphology, as
well as survival of cultured cells, is being assessed using overexpression
and antisense-knockout methodologies. Generation of null mutations in mice
will also be considered depending on the outcome of the cell culture
studies.
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