Chan K W, Sui J L, Vivaudou M, Logothetis D E
Department of Physiology and Biophysics, Mount Sinai School of Medicine, City University of New York, New York, New York 10029, USA.
J Biol Chem. 1997 Mar 7;272(10):6548-55. doi: 10.1074/jbc.272.10.6548.
Heterologous coexpression of recombinant, G protein-gated, inwardly rectifying K+ (GIRK) channel subunits has yielded large currents, severalfold greater than those obtained from expression of the individual subunits. Such current enhancement has been obtained from coexpression of the inactive GIRK1 subunit with the low activity GIRK2-5 subunits in Xenopus oocytes. Using deletion and chimeric constructs, we now report the identification of a C-terminal region unique to GIRK1 and a larger central region of GIRK4 highly homologous to GIRK1, both of which are critical for production of large currents. Chimeras containing these two regions produced homomeric channels, exhibiting currents severalfold greater than those from either wild-type subunit alone. G protein regulation of such chimeric channel currents resembled that of wild-type currents. Green fluorescent protein-tagged channels showed that the amount of chimeric channel expressed on the oocyte cell surface was similar to its wild-type counterpart, suggesting that the enhanced activity was not due to differences in relative levels of expression but rather to the coexistence of the chimeric regions. Single-channel recordings of the active chimeras exhibited patterns of activities with open-time kinetics and conductance characteristics representative of those of GIRK4, indicating that the presence of the GIRK1 C-terminal region caused an increase in the frequency of channel openings without affecting their duration.
重组的G蛋白门控内向整流钾离子（GIRK）通道亚基的异源共表达产生了大电流，比单个亚基表达所获得的电流大几倍。在非洲爪蟾卵母细胞中，无活性的GIRK1亚基与低活性的GIRK2 - 5亚基共表达已获得了这种电流增强。利用缺失和嵌合构建体，我们现在报告鉴定出GIRK1特有的C末端区域以及与GIRK1高度同源的GIRK4的更大的中央区域，这两个区域对于产生大电流都至关重要。包含这两个区域的嵌合体产生了同聚体通道，其电流比单独的任何一种野生型亚基产生的电流大几倍。这种嵌合通道电流的G蛋白调节类似于野生型电流。绿色荧光蛋白标记的通道显示，卵母细胞表面表达的嵌合通道数量与其野生型对应物相似，这表明增强的活性不是由于相对表达水平的差异，而是由于嵌合区域的共存。活性嵌合体的单通道记录显示出具有开放时间动力学和电导特性的活动模式，这些特性代表了GIRK4的特性，表明GIRK1 C末端区域的存在导致通道开放频率增加，而不影响其持续时间。
