Grove A, Tomich J M, Iwamoto T, Montal M
Department of Biology, University of California at San Diego, La Jolla 92093-0357.
Protein Sci. 1993 Nov;2(11):1918-30. doi: 10.1002/pro.5560021113.
To identify sequence-specific motifs associated with the formation of an ionic pore, we systematically evaluated the channel-forming activity of synthetic peptides with sequence of predicted transmembrane segments of the voltage-gated calcium channel. The amino acid sequence of voltage-gated, dihydropyridine (DHP)-sensitive calcium channels suggests the presence in each of four homologous repeats (I-IV) of six segments (S1-S6) predicted to form membrane-spanning, alpha-helical structures. Only peptides representing amphipathic segments S2 or S3 form channels in lipid bilayers. To generate a functional calcium channel based on a four-helix bundle motif, four-helix bundle proteins representing IVS2 (T4CaIVS2) or IVS3 (T4CaIVS3) were synthesized. Both proteins form cation-selective channels, but with distinct characteristics: the single-channel conductance in 50 mM BaCl2 is 3 pS and 10 pS. For T4CaIVS3, the conductance saturates with increasing concentration of divalent cation. The dissociation constants for Ba2+, Ca2+, and Sr2+ are 13.6 mM, 17.7 mM, and 15.0 mM, respectively. The conductance of T4CaIVS2 does not saturate up to 150 mM salt. Whereas T4CaIVS3 is blocked by microM Ca2+ and Cd2+, T4CaIVS2 is not blocked by divalent cations. Only T4CaIVS3 is modulated by enantiomers of the DHP derivative BayK 8644, demonstrating sequence requirement for specific drug action. Thus, only T4CaIVS3 exhibits pore properties characteristic also of authentic calcium channels. The designed functional calcium channel may provide insights into fundamental mechanisms of ionic permeation and drug action, information that may in turn further our understanding of molecular determinants underlying authentic pore structures.
为了识别与离子孔形成相关的序列特异性基序，我们系统地评估了具有电压门控钙通道预测跨膜片段序列的合成肽的通道形成活性。电压门控二氢吡啶（DHP）敏感钙通道的氨基酸序列表明，在四个同源重复序列（I-IV）的每一个中都存在六个片段（S1-S6），这些片段预计会形成跨膜的α螺旋结构。只有代表两亲性片段S2或S3的肽能在脂质双层中形成通道。为了基于四螺旋束基序生成功能性钙通道，合成了代表IVS2（T4CaIVS2）或IVS3（T4CaIVS3）的四螺旋束蛋白。这两种蛋白都形成阳离子选择性通道，但具有不同的特性：在50 mM BaCl2中的单通道电导分别为3 pS和10 pS。对于T4CaIVS3，电导随着二价阳离子浓度的增加而饱和。Ba2+、Ca2+和Sr2+的解离常数分别为13.6 mM、17.7 mM和15.0 mM。T4CaIVS2的电导在高达150 mM盐的情况下都不饱和。虽然T4CaIVS3被 microM Ca2+和Cd2+阻断，但T4CaIVS2不被二价阳离子阻断。只有T4CaIVS3受到DHP衍生物BayK 8644对映体的调节，这表明特定药物作用存在序列要求。因此，只有T4CaIVS3表现出真正钙通道也具有的孔特性。设计的功能性钙通道可能为离子渗透和药物作用的基本机制提供见解，这些信息反过来可能进一步加深我们对真正孔结构潜在分子决定因素的理解。
