Alternative splicing of large-conductance calcium- and voltage-activated potassium (BK) channels

Large conductance calcium- and voltage- activated potassium (BK) channels perform critical and diverse roles including regulation of action potential repolarisation and hyperpolarisation, potassium secretion and neurotransmitter release. Extensive premRNA splicing from the single gene encoding the pore- forming a-subunit provides a mechanism to generate functional diversity of BK channels. Inclusion of different alternatively spliced exons may modify functional properties of BK channels; however the functional role and tissue distribution of different splice variants is largely unknown. The aim of this thesis was to test the hypotheses that: i) alternative splicing may control subcellular localisation of BK channel a-subunits and ii) splice variants are differentially expressed in tissues, using the murine BK channel as the model system. To address whether alternative splice variants may be trafficked specifically to different subcellular compartments, epitope- tagged BK channel splice variants were expressed in mammalian epithelial and endocrine cells. STREX and ZERO variants, in contrast to splice variant Ae23 that is C-terminally truncated, efficiently trafficked to the plasma membrane. Furthermore, splice variants can heteromultimerise in vivo. To investigate tissue specific distribution of splice variants, fluorogenic real time quantitative PCR assays were developed for five known BK channel alternative splice variants- ZERO, e20 (IYF), e21 (STREX), e22, and Ae23 at site C2 of splicing, and used to profile: i) the expression of these splice variants across various tissues in the adult mouse; ii) changes in ZERO and STREX variant expression in the mouse central nervous system during development and iii) STREX variant splicing in steroid responsive tissues of the stress axis. Splice variant expression patterns were distinct in different tissues with, for example, STREX expressed most highly in endocrine tissues and e22 in embryonic tissue. STREX variant expression was significantly reduced in the CNS across the period from embryo day 13 to postnatal day 35, possibly reflecting changes in cell excitability as development progresses and activity- dependent patterning of the CNS is completed. No significant changes in STREX expression were seen under various stress paradigms in adult mice. These data suggest that alternative pre-mRNA splicing is an important determinant of subcellular localisation and that tissues dynamically express a unique complement of BK channel splice variants to serve their physiological demand