Abstract
The research in this thesis describes the way in which various properties of the KcsA tetramer can be influenced by intrinsic protein properties as well as by specific lipids. Different types of mutations within KcsA were made and in chapters 2, 3 and 4 their effects on tetramer stability were
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tested in a variety of different lipid systems. In chapter 2 it was found that replacing the inner or outer helix of KcsA by an alanine/leucine stretch or deleting significant parts of the selectivity filter does not prohibit the protein from forming tetramers. This suggested that the C-terminus is a key player in tetramer assembly, whereas the presence of the selectivity filter in combination with interactions between intramembrane segments of the protein, possibly mediated by lipids, are likely to add to the process of maintaining tetramer stability. In chapter 3 the role of lipids was further investigated and it was established that the anionic phospholipid PA has a specific, charge dependent interaction with KcsA, thereby stabilizing the tetramer. The results suggested that the specific charge properties of PA are the key to enhancing stabilization and it was proposed that the positively charged residues R64 and R89 which are thought to be involved in lipid binding, represent the binding partners of PA in tetramer stabilization. This stabilizing effect by PA was not observed in the chimeric channel KcsA-Kv1.3, indicating that the altered loop in this chimera plays a role in the loss of interaction with PA. Since the basic residue R64 in KcsA is mutated into an aspartic acid in KcsA-Kv1.3 and since additionally R64 had been modeled to be part of a specific lipid binding site via electrostatic interactions, it was speculated that possibly this mutation alone could be responsible for the loss of stabilization by PA in KcsA-Kv1.3. To test this, we introduced the R64D mutation in KcsA-WT in chapter 4. Surprisingly, we found that this mutation leads to a significant increase in tetramer stability compared to KcsA-WT. This enhancement of stability is most likely due to a salt bridge that is formed between D64 and R89 of the adjacent monomer, thereby mimicking the electrostatic interactions that otherwise most likely occur through lipid binding. In chapter 5 we compared the functional properties of the different KcsA-R64 mutants and we found that KcsA-R64D has similar functional properties as KcsA-WT, but with one important difference. The KcsA-R64D mutant is functionally less stable compared to KcsA-WT and most channels close within a few minutes after activation without reopening. Together this implies that lipid binding is involved in structural stabilization of the tetramer as well as in allowing opening and closing of the channel, thereby making it likely that these processes are coupled. Based on these findings we propose in chapter 5 that lipids are part of the mechanism that enables KcsA to transform from the open to closed conformation and vice versa.
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