The Effect of Ethylene Glycol on alpha1 Glycine Receptor Func7on in Xenopus Laevis Oocytes – OSCAR Summer 2023 Abstract and Transcript – Fae Jensen

Ethylene glycol is a common environmental contaminant, as it is a primary component of the solu6on 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. Inges6on of ethylene glycol inhibits central nervous system (CNS) func6oning, 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 func6on in a concentra6on dependent manner. This will be done by microinjec6ng Xenopus laevis oocytes (model cells) 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 GlyRα1s. As GlyRs are one of the primary inhibitory neurotransmiPer receptors of the CNS, these recordings will poten6ally iden6fy a method by which CNS func6oning 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 alpha1 receptors expressed in Xenopus oocytes and whether these effects occur in a concentra6on dependent manner. First I’ll give a brief introduc6on. Ethylene glycol is a widespread environmental contaminant due to its use in fracking solu6on. When ingested, it func6ons as a CNS depressant, though the mechanisms of this CSN 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 conforma6onal 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 nega6ve charge along with it, which can have a hyperpolarizing effect on the cell and overall causes a decreased likelihood for ac6on poten6al genera6on, 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 concentra6on dependent manner upon exposure to ethylene glycol. I propose this will be the case because ethylene glycol and ethanol have a somewhat 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 ethanol is known to allosterically increase glycine receptor response when it binds to this domain. To give you a brief overview of my methods, I first start out by checking my cDNA samples for quality assurance through the use of mass spectrophotometry and gel electrophoresis. 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. This process ensures that I have an adequate and reliable load of GlyRalpha1 RNA to be injected into my oocytes. Following this process, I then fill up a borosilicate glass pipePe with my RNA sample and I 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 oocyte using Two Electrode Voltage Clamping. I use a voltage electrode to read the cell’s internal voltage and a current electrode to measure the current flowing across the membrane by compensa6ng for the change in voltage through the injec6on of a stream of current through a solu6on of potassium chloride. 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. So, now a liPle bit about the perfusion process. The cell is held s6ll and the solu6ons combine in the perfusion system thereby exposing the receptors to the solu6on. First off is just ND96, which is a physiological salt solu6on, this allows me to take baseline recordings of cell func6on prior to any explicit receptor ac6va6on. Following this, I add glycine into the mix at a standard concentra6on of 0.1 mM, allowing me to get baseline recordings of glycine receptor func6on prior to any influence by any other solu6ons. Then, I finally perfuse the cell with incrementally increasing concentra6ons of ethylene glycol, going from 3 uM to 100 uM, 100 uM to 300 uM, and finally 300 uM to 1,000 uM. This allows me to observe how ethylene glycol affects glycine receptor func6on in real 6me and will eventually be used to generate a concentra6on response curve of ethylene glycol’s modula6on of glycine receptor func6on. I have been able to make a total of 8 preliminary recordings for my project, though I have been unable to take my final control recordings due a technological limita6on leaving me unable to clamp the cell’s voltage. Normally the cell’s voltage would be clamped at a set -60 mV, but as you can see here the voltage steadily increased from around -25 mV up to eventually reaching about -10 mV. This causes an inability to read current with defini6ve accuracy, and causes the recordings to be unprotected from the ac6va6on of any voltage-gated ion channels that may be naturally present on the cell. Nonetheless, I have been able to take recordings, though they cannot be read in a defini6ve manner. Here is an example of a control recording I took of an uninjected oocyte. The top lef axis is the current across the membrane in nA, the boPom lef axis is the cell’s voltage in mV, and the x-axis is 6me in minutes. The current depicted here was filtered, meaning that any tonic noise present in the recording was removed. The bidirec6onal changes in amplitude seen across the current recording show mechanical noise. When recording, I no6ced bubbles while changing the solu6on being perfused and this is likely the cause of this mo6on induced noise. Going along the recording from lef to right you can see that I added tags to indicate changes the solu6on being perfused over the cell. The first solu6on I perfused was the basic physiological saline, ND96, resul6ng in no changes in current as expected. The second solu6on was a solu6on of ND96 with 1 mM of glycine added to it. Because the cells were uninjected, and therefore not expressing glycine receptors, the addi6on of glycine did not elicit changes in the cell membrane. This is because without glycine receptors, there were no ion channel pores to be opened and therefore no change in current across the membrane. Afer this, a solu6on of ND96 with glycine with the addi6on of 30 uM ethylene glycol was perfused. No change in current was seen in response to ethylene glycol. This was a good result for my controls, as it showed that ethylene glycol does not interact with proteins that alter conductance on the cell membrane independent of glycine receptor expression. I then incrementally increased the concentra6on of ethylene glycol being perfused over the cell, un6l I eventually reached 1,000 uM. As expected, no changes in current were seen as concentra6on increased. I also took control readings from cells that I had microinjected with water. This was done in order to obtain a baseline for my skill in microinjec6ng, as well as to ensure that cells were able to properly heal during incuba6on. As you can see, no change in current was seen when glycine nor ethylene glycol was perfused. This is because the cells were not microinjected with GlyRalpha1 RNA and therefore did not express glycine receptors. It was good to see that the cell did not experience a change in current between solu6ons, meaning that the cell was able to properly heal and there was no con6nuity between the extracellular fluid and the cell’s contents. It is currently understood how ethylene glycol results in renal failure, though it is s6ll unknown how ethylene glycol results in central nervous system depression, as my project is helping to decipher. Ethylene glycol is first broken down by alcohol dehydrogenase, the same enzyme that breaks down ethanol. When ethanol is broken down by the enzyme, it is then converted into acetaldehyde, which can then be broken down into its rela6vely harmless cons6tuents. When ethylene glycol is broken down though, it is converted into glycoaldehyde, which is then broken down into glycolic acid, glyoxylic acid, and then alpha-hydroxy-beta-ketoadipic acid, oxalic acid, and glycine. It is important to note though that because glycine does not cross the blood brain barrier, this is by no means an indirect mechanism of the central nervous system depression caused by ethylene glycol inges6on. This oxalic acid, though, is going to interact with calcium in the liver to cause the forma6on of calcium oxalate. The calcium oxalate is then going to cause renal tubular necrosis and the calcium absorbed through its synthesis is going to cause hypocalcemia in the liver. As you can see here, the mechanism by which ethylene glycol results in central nervous system depression is yet unknown. My project will address this gap and poten6ally elucidate one of the mechanisms by which the central nervous system depression seen in pa6ents occurs. Here are my references. So, finally I would like to give a big thanks to Dr. Herin for her con6nuous support and mentorship throughout this project, and I would like to thank the OSCAR program for providing me funding in the form of a URSP grant. Thank you!