Park C S, Miller C
Howard Hughes Medical Institute, Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
Neuron. 1992 Aug;9(2):307-13. doi: 10.1016/0896-6273(92)90169-e.
Charybdotoxin (CTX) blocks high conductance Ca(2+)-activated K+ channels by binding to a receptor site in the externally facing "mouth." Toxin bound to the channel can be destabilized from its site by K+ entering the channel from the opposite, internal, solution. By analyzing point mutants of CTX expressed in E. coli, assayed with single Ca(2+)-activated K+ channels reconstituted into planar lipid bilayers, we show that a single positively charged residue of the peptide, Lys-27, wholly mediates this interaction of K+ with CTX. If position 27 carries a positively charged residue, internal K+ accelerates the dissociation rate of CTX in a voltage-dependent manner; however, if a neutral Asn or Gln is substituted at this position, the dissociation rate is completely insensitive to either internal K+ or applied voltage. Position 27 is unique in this respect; charge-neutral substitutions made at other positions fail to eliminate the K+ destabilization phenomenon. The results argue that CTX bound to the channel positions Lys-27 physically close to a K(+)-specific binding site on the external end of the conduction pathway and that a K+ ion occupying this site destabilizes CTX via direct electrostatic repulsion with the epsilon-amino group of Lys-27.
沙蚕毒素（CTX）通过与外向“口部”的受体位点结合来阻断高电导钙激活钾通道。从相反的内部溶液进入通道的钾离子可使结合在通道上的毒素从其位点上不稳定下来。通过分析在大肠杆菌中表达的CTX点突变体，并利用重构到平面脂质双分子层中的单个钙激活钾通道进行检测，我们发现该肽的单个带正电荷残基赖氨酸-27完全介导了钾离子与CTX的这种相互作用。如果第27位带有带正电荷的残基，内部钾离子会以电压依赖的方式加速CTX的解离速率；然而，如果在该位置取代为中性的天冬酰胺或谷氨酰胺，解离速率对内部钾离子或施加的电压则完全不敏感。第27位在这方面是独特的；在其他位置进行的电荷中性取代并不能消除钾离子使毒素不稳定的现象。结果表明，结合在通道上的CTX将赖氨酸-27定位在传导途径外端的钾离子特异性结合位点附近，占据该位点的钾离子通过与赖氨酸-27的ε-氨基直接静电排斥使CTX不稳定。
