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.