Developmental expression of the KCa2.3 channel subunit in the central nervous system and unravelling the molecular basis of the sAHP current regulation

Neuronal calcium-activated potassium currents are intimately involved in the regulation of cellular membrane potential. Different currents have been identified with diverse pharmacological and physiological properties and in many cases the molecular correlates of the currents have been identified. This thesis will seek to provide information about the molecular biology of two distinct Ca2+-activated K+ currents. In this study the distribution of the small conductance Ca2+-activated K+ channel SK3\alpha-subunit in rat brain during development will be described. Firstly, a polyclonal anti-CSK3 antibody was developed and affinity purified. Secondly, this antibody along with a commercial polyclonal antibody against the N-terminus of SK3 have been used to assess the developmental expression pattern of SK3 at five distinct post-natal time points (P1, P3, P6, P12 and P25). The distribution and level of SK3 expression remains constant throughout development and is in accordance with the pattern previously described for adult rat brain. SK3 is highly expressed in many different neuronal populations but of most interest are the high levels of expression seen in monoaminergic regions such as the substantia nigra pars compacta, locus coeruleus and the dorsal raphe nucleus, regions that have been examined in detail here. The slow afterhyperpolarisation is a Ca2+-activated potassium current present in a restricted number of cell types, chiefly the pyramidal cells of the hippocampus and neocortex. A study recently published identified the neuronal calcium sensor hippocalcin, whose expression is also limited to these cell types, as a key molecule responsible for gating the calcium activation of this current (Tzingounis et al. 2007). I have undertaken a yeast two-hybrid screen to identify potential interacting partners of hippocalcin in an attempt to elucidate the molecular pathway underlying the sAHP. This thesis will present the novel interaction between hippocalcin and the Mitogen- Activated Protein Kinase Kinase Kinase (MAP3K), Mixed Lineage Kinase 2 (MLK2 or MAP3K10)