Imredy J P, Chen C, MacKinnon R
Howard Hughes Medical Institute, Laboratory of Molecular Neurobiology and Biophysics, Rockefeller University, New York 10021, USA.
Biochemistry. 1998 Oct 20;37(42):14867-74. doi: 10.1021/bi980929k.
Mamba snake dendrotoxins have been used extensively in biochemical and physiological studies of K+ channels of the brain. Their known targets of inhibition have been limited to the family of voltage-gated K+ channels. We report the isolation of a dendrotoxin inhibitor of ROMK1, a channel belonging to the inward rectifier family of K+ channels. The inhibitory activity, fractionated to purity with FPLC and HPLC, is identical to a previously identified delta-dendrotoxin. To verify that delta-dendrotoxin blocks ROMK1 channels, a cDNA encoding the toxin was synthesized and recombinant toxin expressed in Escherichia coli. Electrophysiological recordings reveal that recombinant delta-dendrotoxin has a half-maximal inhibition constant (Kd) of 150 nM when applied to ROMK1 channels expressed in Xenopus laevis oocytes. That the delta-dendrotoxin binding site exists on separate K+ channel classes is shown by its high affinity for two of the voltage-gated family of channels, Kv1.1 (Kd < 0.1 nM) and Kv1.6 (Kd = 23 nM). Single amino acid substitutions in ROMK1 indicate that delta-dendrotoxin binds to the pore region of ROMK1 even though it does not completely block conduction through the pore. These results suggest that dendrotoxins inhibit K+ channels by recognizing the structurally conserved pore region of these channels.
曼巴蛇树突毒素已被广泛应用于大脑钾离子通道的生化和生理学研究。其已知的抑制靶点仅限于电压门控钾离子通道家族。我们报告了一种ROMK1（一种属于内向整流钾离子通道家族的通道）的树突毒素抑制剂的分离。通过快速蛋白质液相色谱（FPLC）和高效液相色谱（HPLC）将抑制活性分离至纯品，其与先前鉴定的δ-树突毒素相同。为了验证δ-树突毒素阻断ROMK1通道，合成了编码该毒素的cDNA，并在大肠杆菌中表达重组毒素。电生理记录显示，当将重组δ-树突毒素应用于非洲爪蟾卵母细胞中表达的ROMK1通道时，其半数最大抑制常数（Kd）为150 nM。δ-树突毒素对电压门控通道家族中的两个通道Kv1.1（Kd < 0.1 nM）和Kv1.6（Kd = 23 nM）具有高亲和力，这表明δ-树突毒素结合位点存在于不同的钾离子通道类别上。ROMK1中的单个氨基酸取代表明，δ-树突毒素结合到ROMK1的孔区域，尽管它不能完全阻断通过孔的传导。这些结果表明，树突毒素通过识别这些通道结构保守的孔区域来抑制钾离子通道。
