A study of Kv channel dynamics using a fluorescent unnatural amino acid

Proteins are the nature’s molecular nanomachines and their diverse set of functions are crucial to the workings of all cellular mechanisms. A great part of fundamental research today is therefore focusing on the elucidation of protein structure and function. The general objective of this thesis is to expand the understanding of voltage-gated potassium (KV) channels, a group of proteins which governs electrical signalling in various physiological aspects, in particular the propagation of action potentials in the central nervous system and in muscle cells.  In the pursuit of obtaining dynamic information of KV channels, the focus in this thesis has been on exploring the applicability of a fluorescent unnatural amino acid (Anap) as a molecular tool to study protein conformational changes using voltage clamp fluorometry (VCF). The advantage of the Anap-VCF technique over traditional post-translational fluorescence labeling techniques is that there are no restrictions regarding the choice on the site of interest. Anap is genetically incorporated into key regions in the Shaker KV channel by using the amber stop codon suppression technique using an orthogonal tRNA/synthetase pair, and Xenopus laevis oocytes are used as expression system. The first project involves direct comparison of both ends of the voltage sensor (VS) and is made possible by two-color VCF, in which an external cysteine is labeled with TMR (tetramethylrhodamine) while Anap is incorporated on the intracellular side (chapter 3). We found that the intracellular part of VS activates together with the external part. Moreover, it is found that the intracellular S6 gates open in a sequential two-step transition. Next, to investigate electromechanical coupling, Anap was inserted into both ends of the S4-S5 linker (S4-S5L, chapter 4). Two-color VCF experiments demonstrates that the N-terminal part of S4-S5L moves with the VS. On the other hand, the movement of the C-terminal part of the linker is delayed with respect to the VS. The findings support a model for electromechanical coupling in which energy is stored in the middle of the S4-S5L, and not in the C- or N-termini. Moreover, it is found that the S4-S5L undergoes both independent and cooperative movements – a finding which agrees with a pivotal role of the S4-S5 linker in energy transfer from the VS to the pore. In a third project, Anap was inserted into various positions on the flexible and cytosolic N-terminus to probe the movement during N-type inactivation (chapter 5). VCF data show that the hydrophilic chain region of the inactivation particle (IP) undergoes a motion which is closely related to inactivation but is not involved in the final step of pore block. The hydrophobic tip of the IP, on the other hand, undergoes an additional motion which is sensitive to the state of the pore, suggesting that it causes pore block. The findings support a model for inactivation as a sequential step mechanism of at least two transitions. Finally, a fourth project stands out from the general objective of the thesis. It is demonstrated that channels with N-terminal stop codons still express despite the absence of Anap (chapter 6). This leak expression is caused by translation reinitiation at downstream non-canonical start codons and can be reduced by removing the start codons. The findings highlight the importance of control experiments when using unnatural amino acids.

Keyphrases: Anap, Kv channels, voltage clamp fluorometry, VCF, Unnatural amino acids, UAA