CELLULAR AND MOLECULAR BIOLOGY OF RENAL SODIUM CHANNELS

Dartmouth College, Hanover NH

Investigators

Linked publications & trials

Abstract

DESCRIPTION: The long term objective of the investigator's laboratory is to understand the role of epithelial sodium (Na) channels (ENaC) in regulating Na homeostasis and blood pressure in normal and disease states including Liddle's syndrome and Type 1 pseudohypoaldosteronism (PHA1). In Liddle's syndrome, mutations in the b and g subunits of ENaC lead to a gain of ENaC function resulting in hyperabsorption of Na and hypertension. PHA1 is a salt wasting syndrome caused by loss of function mutations in a-, b-, and g-ENaC. Although mutations in ENaC subunits have been implicated in Liddle's syndrome and PHA1, the mechanisms involved in aberrant Na absorption have not been elucidated. Moreover, although ADH stimulates Na absorption across the renal cortical collecting duct (CCD), the mechanisms involved have not been elucidated. The investigator postulates that trafficking of ENaC subunits is disrupted in Liddle's syndrome and PHA1 thereby leading to inappropriate Na absorption. His studies are designed to elucidate the mechanism(s) of ENaC trafficking in the normal state, the role of ADH in ENaC trafficking, and the effects of Liddle's and PHA1 mutations on the trafficking of ENaC subunits. The specific aims include: (1) Developing a living cell model in which to visualize the trafficking of a-, b-, and g-ENaC subunits in CCD cells; (2) Elucidating the pathway(s) by which GFP-ENaC subunits traffic from the ER to the plasma membrane; (3) Testing the hypothesis that acute exposure to ADH stimulates the insertion of ENaC subunits into the apical plasma membrane from a sub-plasma membrane pool; and (4) Determining whether Liddle's and PHA1 mutations alter the trafficking of ENaC subunits. To these ends a living cell model of CCD cells will be generated by transfecting mCCD-K1 cells with green fluorescent protein (GFP)-ENaC fusion protein cDNA constructs. GFP is an autofluorescent protein which, when ligated to a specific protein, serves as a probe to study protein trafficking. Confocal microscopy will be used to image GFP-ENaC in living cells and to determine the distribution of GFP-ENaC in the membranes of the ER, Golgi, sub-plasma membrane compartments, and the apical plasma membrane. The living cell model will be used in studies investigating the effects of ADH and Liddle's and PHA1 mutations on GFP-ENaC trafficking. It is anticipated that these studies will provide new insight into the trafficking of ENaC and on the role of ADH in regulating Na absorption by the CCD. Furthermore, it is anticipated that these studies will provide new information on the cellular and molecular mechanisms responsible for defective Na absorption in patients with Liddle's syndrome and PHA1.