Molecular aspects on voltage-sensor movement

Sammanfattning: Voltage-gated ion channels are fundamental for electrical signaling in living cells. They are composed of four subunits, each holding six transmembrane helices, S1-S6. Each subunit contains a voltage-sensor domain, S1-S4, and a pore domain, S5-S6. S4 contains several positively charged amino-acid residues and moves in response to changes in membrane voltage. This movement controls the opening and closing of the channel. The structure of the pore domain is solved and demonstrates principles of channel selectivity. The molecular mechanism of how the voltage sensor regulates the opening of the channel is still under discussion. Several models have been discussed. One of the models is the paddle model where S3b and S4 move together. The second one is the helical-twist where S4 makes a small rotation in order for the channel to open. The third one is the helical-screw model where S4 twists around its axis and moves diagonally towards the extracellular side of the channel.The aim of this PhD project was to study the molecular movement of the voltage sensor in the depolarization-activated Shaker K channel. Cloned channels were expressed in Xenopus laevis oocytes, and investigated with several electrophysiological techniques.1. We show that S4 moves in relation to both S3b and S5. The formation of some disulfide bonds between S4 and neighboring positions, in only the open state, shows that the paddle model cannot be correct. Furthermore, electrostatic and steric effects of residues in S3b suggest that S3b is tilted, with the intracellular part close to S4.2. We show that the relatively Mg-sensitive Shaker K channel is changed into the less Mg-sensitive Kv2.1 K channel with respect to its sensitivity to extracellularly applied Mg2+ by changing the charge of three extracellularly positioned amino acid residues. One of the residues, F425C, mediates its effect through the neighboring residue K427.3. We show that oxaliplatin, an anti-cancer drug, has no effect on the Shaker K channel. It has been suggested that a negatively charged monochloro complex of oxaliplatin is the active substance, and also causes the neurotoxic side effects. Neither this complex shows any effect on the channel.Our experiments point towards the helical-screw model. The other models for voltage-sensor movements are incompatible with the results in this study.