Cell model of a Principal cell. Aldosterone modulates ENaC in these cells by first affecting gene expression through the mineralocorticoid receptor and inducing expression of the Ki-Ras and Sgk signaling proteins, which then signal to ENaC to modulate ion channel activity.

My laboratory uses a number of contemporary methodologies, including electrophysiology, molecular biology, biochemistry, genomics and proteomics, and fluorescence microscopy to investigate regulation of ENaC and aldosterone signaling. We routinely use yeast, bacteria, immortalized cell lines and animals in this regard. Our lab is equipped to perform real-time measurement of ion channel activity and fluorescence imaging, and performs biophysical analysis of ion channel activity and gating, creates point mutations and chimeric proteins and genes, investigates cellular signal transduction, reconstitutes signaling pathways in heterologous systems, and determines the molecular mechanisms modulating gene expression, and uses the power of yeast genetics in addition to immortalized mammalian cells to investigate ENaC targeting and regulation. The pictures below give some idea of the methods we employ.

Targeting ENaC to different cellular locales. Fusion proteins were created by tagging the cytosolic domains of ENaC with GFP. Cellular localization was determined in a heterologous system by overexpressing these chimeric fusion proteins.

Single channel current recordings for ENaC in cell attached patches. A. ENaC activity in the presence of aldosterone is marked by long open states (downward deflections). B. The frequency of long openings in the presence of aldosterone can be decreased by pre-treating cells with inhibitors of Ki-Ras. C. This maneuver also leads to the shortening of openings.

Idealized schematic showing one way that we test the molecular basis for modulation of gene expression by aldosterone. Two reporter constructs are challenged with aldosterone. Only the one containing the aldosterone-responsive DNA segment respond to hormonal treatment with increased expression of luciferase.