The Effect of Ethylene Glycol on Homomeric α1 Glycine Receptor Function in Xenopus Laevis Oocytes

Ethylene glycol is a common environmental contaminant, as it is a primary component of the solution used in fracking. It remains in the soil and groundwater of fracking sites, therefore causing it to have a sustained effect on both human and other biological life in these areas. Ingestion of ethylene glycol inhibits central nervous system (CNS) functioning, though the specific neuronal mechanisms of this depression are currently unknown. To address this gap in knowledge, this project seeks to determine if ethylene glycol modulates glycine receptor function in a concentration dependent manner. This will be done by microinjecting Xenopus Laevis oocytes with RNA of the α1 subunit of glycine receptors (GlyRα1). Once the receptors are expressed, Two Electrode Voltage Clamp (TEVC) electrophysiology will be used to record transmembrane current caused by Cl- influx through the ion channel pore of the receptors. As GlyRs are one of the primary inhibitory neurotransmitter receptors of the CNS, these recordings will potentially identify a method by which CNS functioning is modulated by ethylene glycol.

Hello! I’m Fae Jensen and I am currently studying the effects of ethylene glycol on glycine receptors of the alpha1 subtype in Xenopus Laevis oocytes and I’m doing this in Dr. Herin’s lab.

The purpose of my experiment is to determine the effects of ethylene glycol on homomeric α1 glycine receptors expressed in Xenopus oocytes and whether these effects occur in a concentration-dependent manner.

Ethylene glycol is a widespread environmental contaminant due to its use in fracking solution. When ingested, it functions as a CNS depressant, though the mechanisms of this depression are currently unknown. Because of this, I’m studying its effects on glycine receptors. This is because when glycine binds to the two binding domains on glycine receptors it causes a conformational change in the receptor which allows the ion channel pore to open, thereby allowing Cl- ion influx into the cell. This Cl- influx brings a negative charge along with it, which can have a hyperpolarizing effect on the cell and overall causes a decreased likelihood for action potential generation, thereby causing CNS depression.

In order to do this I’m using Xenopus Laevis oocytes which are the preferred model system for the study of receptor and ion channel physiology due to their size and membrane durability.

My hypothesis is that Cl- influx through the ion channel pore will increase in a concentration-dependent manner upon exposure to ethylene glycol. I propose this will be the case because ethylene glycol and ethanol have a very similar chemical structure, as you can see here, and I believe this will therefore cause ethylene glycol to bind to the same domain on glycine receptors as does ethanol. And because ethanol is known to allosterically increase glycine receptor response when it binds to this domain.

In order to conduct my experiments, I first start out by checking my cDNA samples for quality assurance. I then replicate the DNA sample if need be, and then transcribe it into mRNA so that it can be translated into proteins once injected into the cytoplasm of cells.
Following this process, I then very controlledly inject 0.05 μL of GlyRα1 RNA into each Xenopus laevis oocyte. I allow the oocytes to incubate for 1-3 days to allow for GlyRα1 proteins to be expressed on the membrane.
I then record from the each oocyte using Two Electrode Voltage Clamping. By clamping this voltage, I am then able to record the actual ion currents which are flowing across the membrane through glycine receptors and other ion channels.

While recording I first begin by perfusing plain ND96 over my cell in order to obtain a baseline recording. I then perfuse my cells with 1 mL of each of my experimental solutions, washing the cell out with ND96 between each solution. This is completed in a randomized order and for sets of solutions consisting of a fixed amount of glycine and varied concentrations of ethylene glycol, as well as for solutions consisting of a set amount of ethylene glycol and varied concentrations of glycine. This allows me to observe how ethylene glycol modulates glycine receptor function in real time, as well as determine whether glycine receptors respond normally to increased concentrations of glycine when exposed to ethylene glycol.

Here are the results from my recordings studying the glycine receptor’s response to various concentrations of glycine while in the presence of 300 uM of ethylene glycol, with the concentration of glycine along the x-axis and the average normalized current from five cells on the y-axis.

And here is my concentration response curve for ethylene glycol recorded across five cells. All of these recordings were taken from cells from the same frog so endogenous ion channel expression may affect these results, but as of yet it is looking like ethylene glycol may in fact potentiate the glycinergic current at concentrations of 300 uM and higher.

Here are my references.

So, finally I would like to give a big thanks to Dr. Herin for her continuous support and mentorship throughout this project, and I would like to thank the OSCAR program for providing me funding in the form of URSP grants. Thank you!!