Effects of Aldosterone on the Ion Channels in the Colon

Wenjuan Gu*, Vazhaikkurichi Rajendran and Andrew Nickerson, Department of Biochemistry, West Virginia University, Morgantown, WV, 26505

Field (Broad Category): Biochemistry (Health Sciences)

Student’s Major: Biochemistry 

Water is absorbed through the guts. The colon absorbs about a third amount of water of that of the small intestine. To absorb water, salt (ions, chloride) is absorbed first which pulls water molecules along with it through osmosis. The gradient usually moves from the Lumen side (intestinal side) to the blood side. The question asked in this experiment is based on how Aldosterone, a hormone, changes the chloride ion channels. The original hypothesis is that the channels would be turned off when there is a depletion in sodium. Rats were put on a sodium depleted diet for a week to increase their Aldosterone production. Western blots were used to determine protein expression for chloride ion channels CFTR and TMEM16a. Calcium chelation is used to separate the epithelium from muscle layers. Extracted proteins were then homogenized in RIPA buffer from tissue samples and sonicated. Protein assay was done by using a BCA protein assay kit. Loading dye and a reducing agent called DTT were loaded onto a polyacrylamide gel called Electrophoresis. Next, the product was transferred onto a membrane (PVDF) and blocked membranes in bovine serum albumin. We probed the membranes for CFTR and TMEM16a and for betaactin. Primary antibodies were put against these three categories and secondary antibody to primary which is conjugated to HRP. The resulting membranes were imaged with substrate. HRP will make substrate glow. The protein band intensity with Image J between control and aldosterone CFTR and TMEM16a were not expected (TMEM didn’t show at all). 

Funding: Grant from the National Institutes of Health 

Program/mechanism supporting research/creative efforts: WVU's Research Apprenticeship Program (RAP) & accompanying HONR 297-level course