We first examined whether total BK, and splice variant, mRNA expression levels differed across distinct regions of the adult (postnatal day 35, P35) murine CNS. To compare the relative expression of total BK channel mRNA transcripts between different CNS regions of P35 mice all data were normalised to the expression of the housekeeping gene, ?-actin, in each region. Total BK channel mRNA expression was variable between different regions with the highest levels observed in frontal cortex and entorhinal cortex (Figure 1b). Regions with the lowest levels (< 50% of the level in entorhinal cortex) were cerebellum, medulla and posterior cortex. The spinal cord, midbrain, pons, thalamus, hypothalamus, hippocampus, olfactory bulb and striatum displayed intermediate total BK mRNA levels (Figure 1b).
Differential expression of the STREX and ZERO BK channel splice variant mRNA was also observed across these tissues at P35. The relative proportion of STREX transcripts (expressed as a percentage of total BK mRNA in each CNS region) was low in all regions tested, with levels typically < 10% of total BK mRNA transcripts. However, even across tissues, the relative proportion of STREX transcripts was significantly different (p < 0.05, Kruskal Wallis test with post-hoc Dunn's test compared to entorhinal cortex) with frontal cortex, thalamus and entorhinal cortex displaying the highest proportion of STREX transcripts and the pons and cerebellum displaying the lowest levels (Figure 1c). The proportion of total BK channel transcripts expressing the ZERO variant also varied significantly across tissues. The entorhinal cortex displayed a significantly (p < 0.01, Kruskal Wallis test with post-hoc Dunn's test) greater proportion of ZERO transcripts compared to any other tissue, with 85.6 ± 9.7 % of total BK transcripts encoded by the ZERO variant. (Figure 1c). In the majority of CNS regions, the proportion of ZERO transcripts was greater than 50% of total BK mRNA transcripts with the notable exception of cerebellum, in which 4.5 ± 2.3 % of transcripts encoded for the ZERO variant (Figure 1c). Taken together, these data suggest that the ZERO variant is the predominant transcript in most adult CNS tissues and that the STREX variant is expressed at a significantly lower level at postnatal day 35. A notable exception is the cerebellum, in which both ZERO and STREX transcripts represent < 10% of total transcripts at P35 suggesting that other site C2 splice variants are predominantly expressed in this region.
Developmental control of BK route mRNA splicing on CNS
So you’re able to look at the developmental changes in BK channel mRNA phrase in almost any aspects of the new murine CNS, BK station mRNA expression was quantified during the embryonic days 13, fifteen and 18, and you can postnatal days seven and you will 35. Each muscle, the full BK channel mRNA term at each developmental big date part is actually indicated since a percentage of these at the P35. For each splice variant the definition of at each developmental big date part are shown since the a share of one’s complete BK channel transcripts when you look at the for every tissue.
Tissues away from rhombencephalon, mesencephalon and spinal cord
When you look at the spinal-cord a tiny, but high, escalation in total BK route mRNA expression is actually noticed away from E13 than the P35 (Shape 2a). It was followed closely by a remarkable reduced amount of STREX version term having a beneficial concomitant rise in No variation mRNA phrase (Contour 4a). Equivalent developmental upwards control out-of total BK channel mRNA term is and additionally found in this new midbrain, cerebellum, pons and medulla (Shape 2b–e). Throughout these buildings, significant developmental downregulation out of STREX version term was again noticed between embryonic and you will postnatal values (Contour 4b–e). Within the midbrain this was paralleled by the a serious escalation in Zero variant term whereas when you look at the pons and Sapiosexual dating service you will medulla no high alter anywhere between E13 and you can P35 was in fact observed (Contour 4d 4e). Compared to most other nations, Zero version phrase significantly ent in the cerebellum (Profile 4c).