Regulation of Myometrial Contractility: Defining the Contribution of the MaxiK Potassium Channel and the L- and T-type Calcium Channels

This thesis describes a comprehensive study investigating the roles of the MaxiK potassium channel (KCNMA1), L-Type calcium channel (CACNA1C) and T-Type calcium channel (CACNA1G) in the maintenance of quiescence (relaxed myometrium), the preparation for parturition (non-contracting myometrium) and the regulation of the co-ordinated contractions characteristic of parturition itself (contracting myometrium). The role of these channels was investigated using primary human myometrial cell cultures under relaxed, non-contracting and contracting conditions. Protein studies revealed changes in both the amount and channel isoforms expressed between the different conditions. Protein-protein interaction studies revealed that the KCNMA1 and CACNA1C associated with Caveolin-1, Gαs and β2-Adrenergic Receptor. RNA studies revealed that the different incubation conditions modified expression of total channel mRNA and that of various splice variants. Previous research has demonstrated that the CACNA1C channel C-terminus can function as a transcription factor termed CCAT. Within this thesis inmmunohistochemistry staining and protein localisation studies revealed nuclear localisation of both the CACNA1C and KCNMA1 C-terminii. Therefore, genomic studies were undertaken utilising the ChIP assay, coupled with ChIP sequencing, to study the role of the KCNMA1 channel as a transcription factor. Chip-sequencing data files were then analysed using Galaxy, an open access web-based platform. Peak calling generated 47 peaks, 21 were successfully mapped to known genes, including RB1, JPH2 and MAP3K7. Motif discovery was then undertaken for both the KCNMA1 protein utilising GYM and the successfully mapped peaks using the Panoptic Motif Search Tool. A helix-turn-helix motif was discovered in the C-terminal region of the KCNMA1 protein and ten putative transcription factor binding motifs were discovered within the peak regions. The significance of these findings is discussed.