End of the Year Report

My research focuses on the involvement of two voltage-gated calcium channel mutations in epilepsy. Epilepsy is a neurological disorder characterized by spontaneous seizures that can cause brain damage. In epilepsy, the normal pattern of neuronal activity becomes disturbed, causing convulsions, muscle spasms, and the loss of consciousness. Epilepsy may develop because of an abnormality in brain wiring, an imbalance in neurotransmitters, changes in ion channels, or some combination of these factors.

Ion channels are cell membrane proteins that allow the passage of ions, such as calcium, into or out of the cell, which generates the electrical signals of neural networks. Voltage-gated calcium channels are a type of ion channel, which allows the passage of calcium ions into the cell. They have a pore through which calcium passes, and one or more auxiliary subunits that regulate pore opening and closing. The auxiliary subunit my research focuses on is the β subunit. The β subunit functions in delivering the calcium channel to the cell membrane and regulating activation and inactivation kinetics of the ion channel. The two mutations in the β subunit that I studied are E53K and Q131L. These mutations were found in a cohort of epileptic patients, but not in unaffected individuals.

In order to study how both of these mutations alter β subunit function and thus voltage-gated calcium channel function, site-directed mutagenesis was performed with QuickChange II XL kit to introduce the desired mutation into the wild-type β subunit. Once we obtained the desired mutation, we synthesized RNA. The RNA, along with the RNA for the other necessary subunits, were injected into Xenopus laevis oocytes. The RNA forms into a calcium channel protein and then we recorded currents using the two-electrode voltage clamp (TEVC). This is done by inserting two glass microelectrodes into the oocyte. One electrode applies the voltage to activate the voltage-gated calcium channel and the other electrode records the resulting currents. Interestingly, we found that the E53K mutant channels had significantly decreased current amplitude when compared to the wild-type and the Q131L mutant channels. This means that not enough calcium ions are able to flow into the cell. We hypothesize that the E53K mutation is causing a decrease in current amplitude by altering channel trafficking. This can lead to various problems such as neurotransmitter imbalance, which is one of the causes of epilepsy.

This research can eventually lead to more personalized and specific treatment that will be more beneficial in treating epilepsy than what is currently available. Performing such research really impacted me and made me feel really happy to discover something that no one has discovered before that can eventually help people and society. I presented my research at two conferences, the NEURON Conference and the Eastern Colleges Science Conference (ECSC). In both conferences my research won an award. I won an Honorable Mention for the Suzannah Bliss Tieman Outstanding Poster Award at the NEURON Conference and I won an award for Outstanding Presentation in Genetics/Molecular Biology at ECSC. While I don’t do research to win awards, it made me feel proud to know that my hard work was recognized by the community and made a significant impact on those who I had presented to.

Overall, I really enjoyed this experience and it is one of the highlights of my four years at Pace. I realized how much I enjoyed being in the lab, constantly learning new things, and doing something that is beneficial to society. Even though I plan to go to medical school in hopes to pursue a career in neurosurgery, I want to incorporate doing research as well in my plans for the future. I am very thankful to my research mentor, Dr. Buraei, and to this program for supporting this research and providing me with this valuable experience.

 

Blog #3- Functional Characterization of Two Calcium Channel Mutations Associated with Epilepsy

Since the last blog post, I injected the RNA I obtained for the I354T mutant along with the RNA for α1 and for the α2δ subunits into Xenopus laevis oocytes. It is important to inject the RNA of the other subunits as well because they are necessary for the formation of a calcium channel protein since it is made up of various subunits. We waited three days before we did electrophysiological recordings so that the protein can be synthesized from the RNA. When we performed electrophysiological recordings using the TEVC, we obtained no current recordings. This could be a result of the RNA for the α1, the pore-forming subunit, no longer being good. This interfered with the formation of the calcium channel protein, thus producing no currents when we recorded from the oocytes. We knew this was not an effect of the mutation because we also did not obtain currents from the wild-type channels.

To fix this problem, I had to synthesize RNA for the α1 subunit. It took several tries until I was finally able to obtain RNA. I obtained RNA on my third try, however I obtained a low yield. Because trying to obtain RNA took a lot of time away from this project and slowed it down, I continued and took up one of the projects of a graduated member of the research team. This project is very similar to the project I am working on. It involves two β subunit mutations, E53K and Q131L. These two mutations are also associated with epilepsy. I performed data analysis using Clampfit and Microsoft Excel on recordings that were obtained for these two mutants and wild-type channels. Interestingly, the E53K mutation reduced the current amplitude of mutant channels compared to the wild-type channels. This means it is not allowing much calcium ions to flow through the channel. We did not see any significant difference with the Q131L mutant so I am currently redoing these experiments to see if I will obtain the same result. Along with redoing the experiments for the E53K and Q131L mutants I will continue the project with the I354T mutation and see if I can get it moving along.

Blog #2-Functional Characterization of the Human β2b I354T Mutation Associated with Epilepsy

As of now, we were able to synthesize RNA for both the wild-type and the I354T mutant. However, we obtained a low yield, meaning we obtained a low concentration of RNA. We are now working on how we can optimize the yield in order to obtain a higher concentration of RNA. RNA is very important because it will be injected into the frog oocytes in order to form a calcium channel protein. Once the calcium channel has formed, then we can perform electrophysiological recordings to compare the currents that go through wild-type calcium channels and calcium channels that contain the mutation.

RNA synthesis is a very delicate and long process. It can easily be contaminated by RNases, which is an enzyme that degrades RNA, that are found in the environment and on human skin. We start the process of synthesizing RNA by first linearizing the wild-type and mutant DNA. Once it is linearized, we purify the DNA by using various chemicals such as phenol/chloroform, chloroform, sodium acetate, and ethanol. This is done to make sure that we have pure DNA and that it is not contaminated. Next, we set up the synthesis reaction using reagents such as the T7 polymerase. After the synthesis reaction, we purify the RNA using similar chemicals that we used for the DNA purification, the only difference being that it is at a different pH. Then, we run our samples on an RNA gel to check the yield and to make sure that it has not been contaminated with RNases. We can tell if it has been contaminated because the bands will appear to be smeared or degraded into separate, smaller bands.

We have gone though this process many times until we finally obtained RNA. RNA is very difficult to make because the process is extremely sensitive since it can be so easily contaminated. Now we are thinking of ways to adjust the protocol so that we can obtain a higher yield of RNA. We think that one reason we may have gotten a low yield was due to the fact that during the synthesis reaction the temperature rose from 37°C to almost 40°C. This sudden rise in temperature may have affected the T7 polymerase, since enzymes are very sensitive to temperature, thus causing us to obtain a low yield. In order to prevent this from happening again, we have to make sure that the incubator we use for the synthesis reaction is set to a steady, constant temperature. Another reason why we may have gotten low yield is because we need to add more T7 polymerase to the synthesis reaction.

Functional Characterization of the Human β2b I354T Mutation Associated with Epilepsy

Epilepsy is characterized by spontaneous seizures that can cause brain damage. In epilepsy, the normal pattern of neuronal activity becomes disturbed, causing convulsions, muscle spasms, and loss of consciousness. Epilepsy may develop because of an abnormality in brain wiring, an imbalance in neurotransmitters, changes in ion channels, or some combination of these factors.

Ion channels are cell membrane proteins that allow the passage of ions, such as calcium, into or out of the cell, which generates the electrical signals of neural networks. My research project will focus on the involvement of a mutation in a voltage-gated calcium channel in epilepsy. Voltage-gated calcium channels have a pore through which calcium passes, and one or more auxiliary subunits that regulate pore opening and closing. The auxiliary subunit that my research will focus on is the β subunit. The β subunit functions in delivering the calcium channel to the cell membrane and regulating activation and inactivation kinetics of the ion channel. The mutation in the β subunit that I will be studying is the I354T mutation, which was found in a cohort of epileptic patients, but not unaffected individuals.

In order to study how the I354T mutation alters β subunit function and thus voltage-gated calcium channel function, site-directed mutagenesis was performed with QuickChange II XL kit to introduce the desired mutation into the wild-type β subunit. Now that the desired mutation is obtained, RNA is going to be synthesized for the I354T mutant. The RNA will then be injected into frog oocytes, which allows for the wild-type and mutant β subunit to be studied. Then, two-electrode voltage clamp will be performed by inserting two glass microelectrodes into the oocyte. One electrode applies the voltage to activate the voltage-gated calcium channels while the other electrode records the resulting currents. This is done to compare the functions of the wild-type and mutant subunits.