Electrophysiological analysis of cloned cyclic nucleotide-gated ion channels
Electrophysiological analysis of cloned cyclic nucleotide-gated ion channels
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DOI：
10.1104/pp.010832
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发表时间：
2002-02-01
期刊：
影响因子：
7.4
通讯作者：
Berkowitz, GA
中科院分区：
文献类型：
--
作者：
Leng, Q;Mercier, RW;Berkowitz, GA
对克隆的拟南芥环核苷酸门控离子通道AtCNGC 2和AtCNGC 1以及烟草NtCBP 4进行了电生理研究。这些蛋白质的核苷酸编码序列在非洲爪蟾卵母细胞或HEK 293细胞中表达。在cAMP存在下，使用电流的电压钳分析评价通道特性。AtCNGC 2被证明可以传导K+和其他一价阳离子，但不包括Na+;这种电导率曲线对于任何不具有K+选择性离子通道的选择性过滤器中发现的氨基酸序列的离子通道来说都是独一无二的。cAMP诱发电流在注射AtCNGC 2 cRNA的卵母细胞膜斑中的应用通过将环核苷酸应用于表达这种通道的膜的补丁来证明，环核苷酸对通道的直接激活是该离子通道家族的标志性特征。电压钳研究（双电极配置）表明，AtCNGC 1和NtCBP 4也是环核苷酸门控通道。将cAMP的亲脂性类似物添加到注射有NtCBP 4和AtCNGC 1 cRNA的卵母细胞的灌注浴中诱导向内整流的非失活K+电流。
Electrophysiological studies were conducted on the cloned plant cyclic nucleotide-gated ion channels AtCNGC2 and AtCNGC1 from Arabidopsis, and NtCBP4 from tobacco (Nicotiana tobacum). The nucleotide coding sequences for these proteins were expressed in Xenopus laevis oocytes or HEK 293 cells. Channel characteristics were evaluated using voltage clamp analysis of currents in the presence of cAMP. AtCNGC2 was demonstrated to conduct K+ and other monovalent cations, but exclude Na+; this conductivity profile is unique for any ion channel not possessing the amino acid sequence found in the selectivity filter of K+-selective ion channels. Application of cAMP evoked currents in membrane patches of oocytes injected with AtCNGC2 cRNA. Direct activation of the channel by cyclic nucleotide, demonstrated by application of cyclic nucleotide to patches of membranes expressing such channels, is a hallmark characteristic of this ion channel family. Voltage clamp studies (two-electrode configuration) demonstrated that AtCNGC1 and NtCBP4 are also cyclic nucleotidegated channels. Addition of a lipophilic analog of cAMP to the perfusion bath of oocytes injected with NtCBP4 and AtCNGC1 cRNAs induced inward rectified, noninactivating K+ currents.