| Summary: | 碩士 === 國立臺灣大學 === 生理學研究所 === 91 === Abstract
The Shaker K+ channel cloned from the fruit fly Drosophila is a member of the voltage-dependent K+ channel family. The opening of Shaker K+ channels is associated with movement of ~13 equivalent electronic charge across the membrane field（or ~3.3 eo per subunit since the channel is a tetramer）. Each subunit of the Shaker K+ channel has 6 transmembrane segments（S1-S6）, and several studies have indicated that most of the gating charges are confined to the fourth transmembrane domain（S4）of each subunit. The S4 segment contains several highly conserved positively charge amino acids. In Shaker K+ channels, at least four of these residues contribute to the voltage sensitivity of the channel:（arginines at position 362,365,368,and 371）. Membrane depolarization moves the positively charged residues of the S4 segment outwardly, but the amount of this displacement and its coupling to gating conformational changes is unknown. Using two-electrode voltage-clamp technique, we studied the gating parameters of 4 mutants（L361F、L361Q、L361R、L361K）of the Shaker K+ channel with expression of the mRNA in Xenopus laevis oocytes. Each of the four mutants tested yielded robust K+ currents, and we also removed the N-type inactivation of each mutant channel to analyze activation behavior more precisely. This study showed that mutant L361F behaves almost the same as the wild-type channel in every respect. In L361R mutant, the half-activation voltage（Vh）of the activation curve was shifted by -31mV along the voltage axis, and the shift was even more pronounced (~-76mV) in the inactivation curve. Moreover, the slope of the activation curve slope is six-fold smaller than that in the wild-type channel, but the change in slope is much less obvious in the inactivation curve. The change in the L361K mutant is in general qualitatively similar to those in L361R but quantitatively even more obvious. On the other hand, the degrees of shift in activation and inactivation curves were similar, and slope of both were not significantly changed in the L361Q mutant. Some of the mutations also significantly affect the gating kinetics of the channel. For example, both the macroscopic activation and inactivation rates are remarkably slower and less voltage-dependent in the L361R mutant than in the WT channels. It seems that a positive-charge substitution at position 361 would uncouple inactivation from activation by “creating”
intermediate gating states that could be inactivated (blocked by the inactivation ball) but are not yet conducting. On the other hand, polar but uncharged substitution at position 361 would not create new intermediate states but stabilize the activated conformation of the channel. Because position 361 is located right external to R362 , the first charged amino acid in the S4 voltage sensor, it is plausible that polar or positive charge amino acid at position 361 would interact with some polar groups or countercharges in the“ S4 gating canal ”and alter the movement and/or the position of S4. Our results indicate tightly correlated but ultimately separate control of the activation and the inactivation gates by the S4 movement.
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