This project investigated whether the expression of specific Kcnma1 transcript variants fluctuated across the estrous cycle in the ventral hippocampus of female mice. Kcnma1 encodes the alpha subunit of a large voltage-gated calcium-activated potassium channel. Of the 22 reported RefSeq Kcnma1 transcripts, 6 contain a 174bp STRess-axis regulated EXon (STREX). STREX codes for a 58 amino acid insert between domains S8 and S9 of the Kcnma1 protein (Fig. 1) resulting in a more excitable potassium channel [1,3]. Due to the role of potassium channels in the regulation of action potentials, upregulation of the expression of STREX-containing variants is associated with an overall increase in the excitability of the hippocampus [1,2]. Further, previous studies have shown that expression of STREX-containing variants in the uterine myometrium is regulated by sex hormone levels . To determine whether sex hormone fluctuations are regulating the expression of STREX-containing variants in the ventral hippocampus, I designed RT-PCR primers that would amplify all Kcnma1 transcript variants, yielding different sized products depending on the transcripts amplified (Fig 2).
Figure 1-Kcnma1 protein with the location of the STREX insert.
Figure 2-Table depicting the 4 expected RT-PCR products using the first set of Kcnma1 primers. The STREX-containing variants and the corresponding RT-PCR product are highlighted in red.
Figure 3-RT-PCR results using primers amplifying all Kcnma1 transcripts. Arrows indicate bands with sizes corresponding to the expected products outlined in Figure 2.
Figure 4-RT-PCR results using primers targeting STREX-containing Kcnma1 transcripts (top left), loading control CypA (bottom left), and quantification of results (right). (* = p < 0.05)
Previous work in the Kundakovic lab has demonstrated that the hormonal fluctuations characterizing the estrous cycle are associated with differential gene expression and chromatin accessibility in the ventral hippocampus of female mice. This project aimed to evaluate whether the relative expression of specific mRNA transcript variants fluctuate across the estrous cycle in a hormone-state dependent manner. The target gene, Kcnma1, encodes the alpha subunit of a large voltage-gated calcium-activated potassium channel. Potassium channels function in regulating neuronal excitability through their role in action potentials . Kcnma1 has several transcript variants coding for isoforms with different functional properties . Although it has been shown that certain Kcnma1 transcripts are upregulated in response to sex hormones , the influence of sex hormone fluctuations on Kcnma1 transcription in the brain has yet to be explored. Here I show differential expression of specific Kcnma1 transcripts at various estrous cycle stages in the ventral hippocampus of female mice.
Thanks to Dr. Kundakovic and her lab for the mice used in this experiment and for guidance in estrous cycle stage determination, dissection of the ventral hippocampus, and RNA isolation. Thanks to Tony and Faaria for their enormous help in the molecular procedures performed in this project. Last, thanks to Dr. Rubin whose guidance and patience made this project possible.
|This document was last modified 05/15/2018.|
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|This is a project of the Biology Department of Fordham University