Effects of point mutations on the block of SK3 small-conductance calcium-activated potassium channels expressed in mammalian cell lines

Small conductance calcium-activated K+ channels (SK channels) form a subfamily of K+ -selective, volt age-insensitive channels that have been recently cloned. This thesis is concerned primarily with an investigation of the pharmacology of these channels. Understanding their pharmacology is of potential clinical importance since SK channels participate in diverse physiological roles, for example, generating neuronal afterhyperpolarizations (that set tonic firing frequencies) and regulating smooth muscle contraction. This thesis consists of two parts. The first is concerned with expressing and establishing recording conditions for rSK3 in mammalian cell lines. This was made difficult because of channel run up and run down. However, some progress was made towards stabilizing channels through MgATP regulation and this allowed progress towards the second part (the major part) of this thesis; a functional characterization of site-directed point mutants that were created to better understand the pharmacology of these channels. First, two mutations were made to examine similarities between the pore structures of the KcsA and Shaker channels and the SK channels. The first of these mutants would be predicted to increase Tetraethylammonium (TEA) affinity to the sub-millimolar range, and the second to provide increased sensitivity to charybdotoxin (CTX) block. Both these predictions were fulfilled suggesting that the KcsA/Shaker pore structure can provide a reasonable model for the SK channel pore. However, an important difference was identified in the TEA mutant, indicating that although the channels are similar, they are not identical. Three UCL compounds were then studied; UCL 1848, UCL 1684, and UCL 1530 with six channel mutants. The effects of these mutations on blocker affinity provided the basis of a map of the channel residues interacting with UCL compounds and establishes that these blockers bind in the channel pore region. Further, experiments co-expressing wildtype (WT) and mutant subunits demonstrate that UCL compounds do not require all four sensitive subunits for block, suggesting an asymmetric interaction with the channel outer pore. Finally, some work has also been done to define the possible assembly patterns of SK subunits in forming heteromeric channels. Evidence is presented that SKI and SK3 can co-assemble. Overall, this thesis provides a starting point for understanding the pharmacology of small molecule SK channel blockers at the molecular level.