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D'hoedt, Dieter (2005): Structure-function analyses of small-conductance, calcium-activated potassium channels. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Ion channels are integral membrane proteins present in all cells. They are highly selective and assure a high rate for ions down their electrochemical gradient. In particular, small-conductance calcium-activated potassium channels (SK) are conducting potassium ions and are activated by binding of calcium ions to calmodulin, which is constitutively bound to the carboxy-terminus of each SK channel -subunit. Until now, only three SK channel subunits have been cloned, SK1, SK2 and SK3. Sequence alignment shows that the transmembrane and pore regions are highly conserved, while a high grade of divergence is observed in the amino- and carboxy-termini of the three subunits. In order to determine the expression of the different SK channel subtypes, pharmacological tols such as apamin and d-tubocurarine have been widely used. In this work, I show the characterization of a novel toxin, tamapin, isolated from the scorpion Mesobuths tamulus, which targets SK channels. Our experiments show that this toxin is more potent in blocking SK2 channels than apamin. Furthermore, tamapin only blocked the SK1 and SK3 channels at higher concentrations, with higher efficiency to block SK3 than SK1. Therefore, tamapin should be a good pharmacological tool to determine the molecular composition of native SK channels underlying calcium-activated potassium currents in various tissues. Secondly, I determined the molecular mechanism that prevents the formation of functional SK1 channels cloned from the rat brain (rSK1). Until now, little information was available on the rSK1 channels. rSK1 shows highly sequence identity (84%) with the human homologue, hSK1. hSK1 subunits form functional potassium channels that are blocked by apamin and d-tubocurarine. However, when I expressed rSK1 in HEK-293 cells no potassium currents above background were observed, although immunofluorescence experiments using a specific antibody against the rSK1 protein showed expression of the channel. I generated rSK1 core chimeras in which I exchanged the amino-and/carboxy-terminus with the same region of rSK2 or hSK1. Exchange of amino-and carboxy-terminus or only of the carboxy-terminus resulted in the formation of functional potassium channels. Furthermore, I used these functional chimeras to determine the toxin sensitivity of rSK1 for apamin and d-tubocurarine. Surprisingly, when these blockers wre applied, no sensitivity was observed, although hSK1 and rSK1 show a complete sequence identity in the pore region, which is suggested to contain the binding site for apamin. Finally, I characterized a novel splice variant of the calcium-activated potassium channel subunit rSK2, referred to as rSK2-860. The rSK2-860 cDNA codes for a protein which is 275 amino acids longer at the amino-terminus when compared with originally cloned rSK2 subunit. Transfection of rSK2-860 in different cell lines resulted in a surprising expression pattern of the protein. Th protein formed small clusters around the cell nucleus, but no membrane stain could be observed. This data shows that the additional 275 amino acid-long stretch at the amino-terminus is responsible for retention and clustering of rSK2-860 protein. In order to narrow down the region responsible for this phenotype, I generated truncated proteins. This resulted in the isolation of an 100 amino acid-long region that seems to be responsible for the retention and clustering of rSK2-860 channels. Further truncations and deletions could help us to find the exact signal which is responsible for this characteristic behavior of the rSK2-860 protein.