Hohaus A, Poteser M, Romanin C, Klugbauer N, Hofmann F, Morano I, Haase H, Groschner K
Max-Delbrück-Centrum für Molekulare Medizin, D-13092 Berlin, Germany.
Biochem J. 2000 Jun 15;348 Pt 3(Pt 3):657-65.
Modulation of the smooth-muscle Ca(2+) channel alpha1C-b subunit by the auxiliary beta2a subunit was studied in the HEK 293 (cell line from human embryonic kidney cells) expression system. In addition, we tested whether the alpha1-beta interaction in functional channels is sensitive to an 18-amino-acid synthetic peptide that corresponds to the sequence of the defined major interaction domain in the cytoplasmic I-II linker of alpha1C (AID-peptide). Ca(2+) channels derived by co-expression of alpha1C-b and beta2a subunits exhibited an about 3-fold higher open probability (P(o)) than alpha1C-b channels. High-P(o) gating of alpha1C-b.beta2a channels was associated with the occurrence of long-lasting channel openings [mean open time (tau)>10 ms] which were rarely observed in alpha1C-b channels. Modulation of fast gating by the beta2a subunit persisted in the cell-free, inside-out recording configuration. Biochemical experiments showed that the AID-peptide binds with appreciable affinity to beta2 subunits of native Ca(2+) channels. Binding of the beta2 protein to immobilized AID-peptide was specifically inhibited (K(i) of 100 nM) by preincubation with free (uncoupled) AID-peptide, but not by a corresponding scrambled peptide. Administration of the AID-peptide (10 microM) to the cytoplasmic side of inside-out patches induced a substantial reduction of P(o) of alpha1C-b.beta2a channels. The scrambled control peptide failed to affect alpha1C-b. beta2a channels, and the AID-peptide (10 microM) did not modify alpha1C-b channel function in the absence of expressed beta2a subunit. Our results demonstrate that the beta2a subunit controls fast gating of alpha1C-b channels, and suggest the alpha1-beta interaction domain in the cytoplasmic I-II linker of alpha1C (AID) as a possible target of modulation of the channel. Moreover, our data are consistent with a model of alpha1-beta interaction that is based on multiple interaction sites, including AID as a determinant of the affinity of the alpha1-beta interaction.
在HEK 293（源自人胚胎肾细胞的细胞系）表达系统中研究了辅助β2a亚基对平滑肌Ca(2+)通道α1C - b亚基的调节作用。此外，我们测试了功能性通道中的α1 - β相互作用是否对一种18个氨基酸的合成肽敏感，该肽对应于α1C胞质I - II连接区中确定的主要相互作用域的序列（AID肽）。由α1C - b和β2a亚基共表达产生的Ca(2+)通道的开放概率（P(o)）比α1C - b通道高约3倍。α1C - b.β2a通道的高P(o)门控与长时程通道开放的出现有关[平均开放时间（τ）>10毫秒]，而在α1C - b通道中很少观察到这种情况。β2a亚基对快速门控的调节在无细胞的内向外记录模式中持续存在。生化实验表明，AID肽与天然Ca(2+)通道的β2亚基有明显的亲和力结合。β2蛋白与固定化AID肽的结合可通过与游离（未偶联）AID肽预孵育而被特异性抑制（抑制常数K(i)为100 nM），但相应的乱序肽则无此作用。将AID肽（10 μM）施加到内向外膜片的胞质侧会导致α1C - b.β2a通道的P(o)大幅降低。乱序对照肽未能影响α1C - b.β2a通道，并且在没有表达β2a亚基的情况下，AID肽（10 μM）也未改变α1C - b通道的功能。我们的结果表明，β2a亚基控制α1C - b通道的快速门控，并提示α1C胞质I - II连接区中的α1 - β相互作用域（AID）可能是通道调节的一个靶点。此外，我们的数据与基于多个相互作用位点的α1 - β相互作用模型一致，其中AID是α1 - β相互作用亲和力的决定因素。
