Voltage sensor movements in shaker and HCN channels

Sammanfattning: Voltage-gated ion channels are crucial for electrical signaling in living organisms. They are composed of four similar subunits with six transmembrane ?-helices, S1-S6. Each subunit contains a voltage-sensing domain, S1-S4, and an ion-conducting pore domain, S5-S6. S4 contains several positively charged residues and moves in response to changes in the transmembrane voltage. This movement controls the opening and the closing of the ionconducting pore. The structure of the pore domain of potassium channels has been solved at atomic resolution and demonstrates principles of channel selectivity and opening. The molecular mechanisms of how the voltage sensor regulates the opening of the pore are not known. The aim of the present PhD project was to study the molecular movements of the voltage sensor in the depolarization-activated Shaker channel and in hyperpolarizationactivated cyclic-nucleotide-gated HCN channels. Cloned ion channels were expressed in Xenopus leavis oocytes, and investigated with several electrophysiological techniques. 1. We could show that the outermost charged voltage-sensor residue of the Shaker channel, R362, has great electrostatic effect on the extracellular end of S5 in the activated state. It was shown that R362 is located more than 20 A away from a residue 418C in the pore domain in the resting state, and that it moves close to (8 Å) the pore residue during channel activation. 2. The voltage sensor S4 of an HCN channel was shown to move in a similar way as S4 of the Shaker channel with respect to the membrane/protein plane. The exposure of several individually introduced cysteines in S4 to the extra- and intracellular solutions was found to be state dependent. One cysteine in the voltage sensor was found to move across the membrane/protein plane during channel activation. We recorded the gating currents of HCN channels. Modification of a cysteine in several positions of S4 dramatically decreases the gating currents, suggesting that the modification blocks the movement of the voltage sensor. 3. We also found that the voltage dependence of the gating currents in HEN channels depends on preceding voltage pulses, that is, it is history dependent. The voltage dependence of the gating currents was shifted +60 mV when the holding voltage was -80 mV instead of 10 mV. The shift was shown to occur on a physiological time scale of rhythmicity and in a physiological range of voltages. Thus, there is a hysteresis in the voltage dependence of the voltage-sensor movement of HCN channels. Ionic currents display a similar hysteresis. The hysteresis in the voltage dependence was found to be beneficial for rhythmicity in a model of sino-atrial node cells. A comprehensive model of the channel's function was constructed to describe the complex voltage dependence and the kinetic properties of the HCN channel function.