Shaker鉀離子通道S4穿膜區段之位移及其對門閥開關之影響

Shaker 鉀離子通道屬於一種電位開關性鉀離子通道。此種通道是由四個次單元組成，每一個次單元又是由六條穿膜蛋白 ( S1-S6 ) 連接而成。其中，S5-S6組成了通道的孔洞。而S4上因為帶有一些規則排列的鹼性胺基酸，因而被認為可能與感測電場變化有關。當細胞膜去極化時，S4能感測電場變化而移動，進而使得通道孔洞開啟。近年來，關於S4的運動方式的說法主要有三種，第一種：S4的運動主要藉由扭轉來使得孔洞打開，而非位移。第二種：S4以螺旋向上的方式運動。第三種：S4位於離子通道的最外圍且與細胞膜接觸，移動的方式是向上簞吽C此三種模式皆各自有實驗佐證，而本實驗亦欲探討S4的運動及其對通道開關的影響。藉由S3-S4 linker的活動可以幫助了解S4的運動方式。當通道活化時，S4上的帶正電鹼性胺基酸可能會隨著S4的上移而移動到原本S3-S4 linker的位置。意味著在S3-S4 linker的某些位置上可能具有能夠穩定帶正電鹼性胺基酸的環境。於是我們從360開始往前到353做正電胺基酸arginine的突變，發現L358R、L358K與L358Q突變通道的活化、不活化曲線之Vh皆向左移動且活化速率與不活化速率都明顯比正常通道快釵h，而L358F突變通道卻無此情形。因此猜測358位置的厭水特性有某種程度的重要性。當S4處於休息態時，358位置的厭水性胺基酸與其周圍環境的厭水性胺基酸之間的作用力可能具有維持S4在休息態位置的能力。此外，更有趣的是，A359R與M356R突變通道之活化、不活化曲線相對於正常通道都有明顯右移但斜率變化不顯著之情形。且其活化、不活化速率也較正常通道來的慢。推測這兩個位置極可能含有能夠穩定arginine的極性或帶相反電荷的胺基酸存在。由於356及359位置恰好與R362相差了3個及6個胺基酸，故此一發現可能暗示著當細胞膜去極化，S4向上移動時，S4上的第一個鹼性胺基酸R362可能會依序經過359與356的位置。而R365與R368(或R362與R365)也能恰好對應到356與359的位置。亦即S4在運動過程中一共轉動了120或180度，移動6或9個胺基酸的距離。

關鍵字

活化；第四穿膜片段；第三第四穿膜蛋白之連接片段；鉀離子通道；門閥開關

並列摘要

Shaker K+ channels constitute an important group of voltage-gated K+ channels which are composed of four subunits, each of which has six transmembrane segments (S1-S6). S5-S6 form the pore domain. S4 contains regularly spaced basic residues and has been considered as the primary voltage sensor of the channel. There are chiefly three models explaining the S4 movement, namely the twist model (a twist motion of S4 without significant translational movement), the helical screw model (combined translational and rotational movement of S4), and the paddle model (S4 lying at the channel periphery and moving as a unit with S3b). They all emphasize that S4 movement must play an important role in channel gating, but give different molecular pictures for the movement of S4. Because the S3-S4 linker is directly connected to S4, one may try to elucidate the molecular features of S4 movement by manipulation of the S3-S4 linker. We made point mutations in the S3-S4 linker, from I360 which is located external to the outermost positive residue (R362 ) to N353. In L358R mutant channels, the Vh of activation and inactivation curves are leftwardly shifted, and the activation and inactivation rates are much faster as compared to the wild type channels. There are very similar findings in L358Q but not L358F mutant channels. A hydrophobic residue in position 358 thus may play a critical role in stabilization of S4 at its resting position. Most interestingly, the Vh of the activation and inactivation curves are significantly rightwardly shifted in A359R and M356R mutants as compared to the wild type channels.Together with the evidently slower activation and inactivation rates, these findings suggest that some polar or acidic residues exist in the “gating canal” and would favorably inteact whith the basic side chains of the amino acids at positions 359 and 356. Because positions 359 and 356 are 3 and 6 residues apart from R362, it is plausible that the strong stabilization force on arginines at these positions might be designated for R368 and R365 (or R365 and R362), respectively, in the S4 of the activated channel. S4 thus probably moves through the gating canal by 6-9 residues and rotate about 120o-180o with its basic residues coordinated by those polar residues or counter charges located at appropriate positions in the canal at each ratchet step of the movement.

並列關鍵字

S4 movement ； Shaker potassium channel ； activation ； gating ； S3-S4 linker

參考文獻

Aggarwal, S. K., and MacKinnon, R. (1996). Contribution of the S4 segment to gating charge in the Shaker K+ channel. Neuron 16, 1169-1177.

Aziz, Q. H., Partridge, C. J., Munsey, T. S., and Sivaprasadarao, A. (2002). Depolarization induces intersubunit cross-linking in a S4 cysteine mutant of the Shaker potassium channel. J Biol Chem 277, 42719-42725. Epub 42002 Aug 42723.

Baker, O. S., Larsson, H. P., Mannuzzu, L. M., and Isacoff, E. Y. (1998). Three transmembrane conformations and sequence-dependent displacement of the S4 domain in shaker K+ channel gating. Neuron 20, 1283-1294.

Cha, A., Snyder, G. E., Selvin, P. R., and Bezanilla, F. (1999). Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy. Nature 402, 809-813.

Doyle, D. A., Morais Cabral, J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998). The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280, 69-77.

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Shaker鉀離子通道S4穿膜區段之位移及其對門閥開關之影響

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