Hanner M, Vianna-Jorge R, Kamassah A, Schmalhofer W A, Knaus H G, Kaczorowski G J, Garcia M L
Department of Membrane Biochemistry and Biophysics, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
J Biol Chem. 1998 Jun 26;273(26):16289-96. doi: 10.1074/jbc.273.26.16289.
Coexpression of alpha and beta subunits of the high conductance Ca2+-activated K+ (maxi-K) channel leads to a 50-fold increase in the affinity for 125I-charybdotoxin (125I-ChTX) as compared with when the alpha subunit is expressed alone (Hanner, M., Schmalhofer, W. A., Munujos, P., Knaus, H.-G., Kaczorowski, G. J., and Garcia, M. L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2853-2858). To identify those residues in the beta subunit that are responsible for this change in binding affinity, Ala scanning mutagenesis was carried out along the extracellular loop of beta, and the resulting effects on 125I-ChTX binding were determined after coexpression with the alpha subunit. Mutagenesis of each of the four Cys residues present in the loop causes a large reduction in toxin binding affinity, suggesting that these residues could be forming disulfide bridges. The existence of two disulfide bridges in the extracellular loop of beta was demonstrated after comparison of reactivities of native beta and single-Cys-mutated subunits to N-biotin-maleimide. Negatively charged residues in the loop of beta, when mutated individually or in combinations, had no effect on toxin binding with the exception of Glu94, whose alteration modifies kinetics of ligand association and dissociation. Further mutagenesis studies targeting individual residues between Cys76 and Cys103 indicate that four positions, Leu90, Tyr91, Thr93, and Glu94 are critical in conferring high affinity 125I-ChTX binding to the alpha.beta subunit complex. Mutations at these positions cause large effects on the kinetics of ligand association and dissociation, but they do not alter the physical interaction of beta with the alpha subunit. All these data, taken together, suggest that the large extracellular loop of the maxi-K channel beta subunit has a restricted conformation. Moreover, they are consistent with the view that four residues appear to be important for inducing an appropriate conformation within the alpha subunit that allows high affinity ChTX binding.
与单独表达α亚基时相比，高电导钙激活钾通道（大电导钾通道）的α亚基和β亚基共表达使对125I - 蝎毒素（125I - ChTX）的亲和力增加了50倍（汉纳，M.，施马尔霍费尔，W. A.，穆努霍斯，P.，克瑙斯，H.-G.，卡佐罗夫斯基，G. J.，以及加西亚，M. L.（1997年）《美国国家科学院院刊》94，2853 - 2858）。为了确定β亚基中负责这种结合亲和力变化的那些残基，沿着β亚基的细胞外环进行丙氨酸扫描诱变，并在与α亚基共表达后确定其对125I - ChTX结合的影响。环中存在的四个半胱氨酸残基中的每一个发生诱变都会导致毒素结合亲和力大幅降低，这表明这些残基可能正在形成二硫键。在比较天然β亚基和单半胱氨酸突变亚基与N - 生物素 - 马来酰亚胺的反应性后，证实了β亚基细胞外环中存在两个二硫键。β亚基环中的带负电荷残基，单独或组合突变时，除了Glu94外对毒素结合没有影响，Glu94的改变会改变配体结合和解离的动力学。针对半胱氨酸76和半胱氨酸103之间的单个残基的进一步诱变研究表明，四个位置，即亮氨酸90、酪氨酸91、苏氨酸93和谷氨酸94对于赋予αβ亚基复合物高亲和力125I - ChTX结合至关重要。这些位置的突变对配体结合和解离的动力学有很大影响，但它们不会改变β亚基与α亚基的物理相互作用。综合所有这些数据表明，大电导钾通道β亚基的大细胞外环具有受限的构象。此外，它们与以下观点一致，即四个残基似乎对于在α亚基内诱导允许高亲和力ChTX结合的合适构象很重要。
