These past few months, Dr. Zafir Buraei and I have been working on the effects of Presenilin on a class of ion channels known as TRP Channels. Studies conducted in the past depicts that Presenilin can also act as a low conductance leak channel for Calcium ions on the Endoplasmic Reticulum. TrpC channels are often activated by the efflux of calcium ions from the endoplasmic reticulum, a cellular structure that serves as storage for intracellular calcium. The efflux of calcium ions that activate TRP channels begin with a whole cascade of G-Protein Coupled Receptors that are activated by applying an agonist (drug) that allows phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid found on the cell membrane along with phospholipase C (PLC) to catalyze the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Calcium ions are than depleted from the ER by the binding of IP3 on IP3 receptors and allowing calcium to flow out activating TRP channels. On the other hand DAG can also activate TRP channels by direct binding.
After obtaining our cDNA’s for Presenilin Wild-type, Presenilin Mutant (M146v), TRPC5, M1R G-Protein Coupled Receptors (Muscarinic acetylcholine receptors); we linerized DNA overnight and made RNA over a 2 day process. Then performing gel electrophoresis, we confirmed if the RNA we had produced was the correct size in base pairs and if it was ready to inject oocytes. Many times the gel electrophoresis was not a success but after a series of troubleshooting steps, we finally were able to generate RNA suitable for frog oocytes. Our next step was going to Columbia University and obtaining oocytes from the African clawed frog: Xenopus Laevis. After performing surgery under anesthesia, we prepared the oocytes by a series of washing steps in OR2 buffer and defoliculating them so its easier to inject and to obtain membrane currents. The last wash consisted of ND96 buffer in which oocytes are incubated and in which recordings are also taken.
We injected various groups into the batch of chosen oocytes such as: (0.5 ul M1R + 1 ul dH20), (0.5 ul TRPC5 + 1 ul dH20) (0.5 ul M1R+ 0.5 ul TRPC5+ 0.5 ul dH20), (0.5 ul M1R+ 0.5 ul TRPC5+ 0.5 ul PresWT), (0.5 ul M1R+ 0.5 ul TRPC5+ 0.5 ul PresM146V). We used 1.5 ul total volume for every group which allowed us to inject about 30 oocytes, in which each oocytes got 50 nL of prepared RNA of those groups. We did recordings using the Two-Electrode Voltage Clamp (TEVC), the third day after injections for which we prepared two extracellular solutions that are used to obtain currents. The solutions were the same except one of them had 40 uM Carbachol added to 40 mL of the extracellular solution as a drug that activates Muscarinic acetylcholine receptors. The solution itself was made of 123 mM NaCl, 2 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES pH adjusted to 7.4 and 0.3 mM Niflumic acid with a osmolarity of 297 osm/L.
The results we obtained proved our hypothesis right, that Presenilin drastically drained TRPC5 currents than the group without Presenilin. Furthermore, as we read in various studies Presenilin M146v has the same effect as the wild type presenilin on oocytes. Our next experiments that will be conducted will utilize different Presenilin mutations that do not have the same effect as wild-type Presenilin. Furthermore, already in progress we will compare the results of these groups to new groups that have Presenilin siRNA, which will knockout the endogenous Presenilin that oocytes are said to already have.
This project allowed me to gain a vast amount of knowledge on TRP channel function along with Presenilin and the entire G protein coupled receptor cascade that in turn activates TRP channels. Also on the other hand this project allowed me to understand and learn how to use the two electrode voltage clamp (TEVC) for oocyte recordings after learning to inject them alongside Dr. Zafir Buraei. I also learned how to carefully analyze and interpret the data that was obtained from TEVC recordings of oocytes. My future goals and progressions with this project is to test and obtain suitable data for other presenilin mutations and using siRNA, which will give us a better insight on TRP channel function alongside Presenilin known to cause Alzheimer’s disease. Our ultimate goal is to shed light on this topic and generate enough data to publish an article.