Ferraro, T.; (2006) Cellular localisation and function of neuronal SK potassium channels. Doctoral thesis , University of London.

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Abstract

In the central nervous system, small conductance calcium-activated potassium channels (SK) are important for the generation of the afterhyperpolarisation that follows single or trains of action potentials. Three SK channels have been cloned (SKI, SK2 and SK3), which are selectively blocked by the venom toxin apamin and present distinct pharmacological properties. The principal aim of this work is to investigate the subcellular distribution of SK2 and SK3 channels in neurones. For this purpose, antibodies directed against specific regions of SK2 and SK3 subunits were raised, purified and characterised. Immunohistochemistry on rat brain sections showed that the SK2 protein is localised in somatic and dendritic structures in various brain regions. SK3 immunoreactivity is mainly associated with fibers, but also with neuronal somata and dendrites. Intense SK3 expression was detected in the substantia nigra pars compacta, the locus coeruleus and the dorsal raphe. In these brain regions, the distribution of the channel was analysed in relation to monoamine- containing neurones, at two different developmental stages. In this work, I also report the characterization of a new SK2 variant, SK2-860, which differs from the SK2 subunit by having an extended amino terminus. Heterologous expression of this variant in primary neurones indicates that the majority of the protein is located in clusters distributed throughout the cytoplasm. Western blot analysis with the SK2 antibody and with a specific antibody for the new variant showed that SK2-860 subunit is expressed in the rat central nervous system. The distribution pattern of the SK2-860 in the rat brain largely overlaps the one observed for SK2. Finally, in the last part of this work, the molecular determinants responsible for the difference in apamin sensitivity among the different members of the SK channel family were investigated. The study showed that the extracellular loop that connects transmembrane segments S3 and S4 of SK channel a subunits significantly contributes to apamin sensitivity of SK2 channels.

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