Department of Physiology, McGill University, Montreal, Quebec, Canada
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

RESEARCH INTERESTS


 

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.
 


NHE molecule


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.
 


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).


NHE1 in cardiac myocytes

   
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.

 

This page was last edited on 21 July, 2004