Liu G X, Derst C, Schlichthörl G, Heinen S, Seebohm G, Brüggemann A, Kummer W, Veh R W, Daut J, Preisig-Müller R
Institut für Normale und Pathologische Physiologie, Marburg University Deutschhausstrasse 2, D-35037 Marburg, Germany.
J Physiol. 2001 Apr 1;532(Pt 1):115-26. doi: 10.1111/j.1469-7793.2001.0115g.x.
The aim of the study was to compare the properties of cloned Kir2 channels with the properties of native rectifier channels in guinea-pig (gp) cardiac muscle. The cDNAs of gpKir2.1, gpKir2.2, gpKir2.3 and gpKir2.4 were obtained by screening a cDNA library from guinea-pig cardiac ventricle. A partial genomic structure of all gpKir2 genes was deduced by comparison of the cDNAs with the nucleotide sequences derived from a guinea-pig genomic library. The cell-specific expression of Kir2 channel subunits was studied in isolated cardiomyocytes using a multi-cell RT-PCR approach. It was found that gpKir2.1, gpKir2.2 and gpKir2.3, but not gpKir2.4, are expressed in cardiomyocytes. Immunocytochemical analysis with polyclonal antibodies showed that expression of Kir2.4 is restricted to neuronal cells in the heart. After transfection in human embryonic kidney cells (HEK293) the mean single-channel conductance with symmetrical K+ was found to be 30.6 pS for gpKir2.1, 40.0 pS for gpKir2.2 and 14.2 pS for Kir2.3. Cell-attached measurements in isolated guinea-pig cardiomyocytes (n = 351) revealed three populations of inwardly rectifying K+ channels with mean conductances of 34.0, 23.8 and 10.7 pS. Expression of the gpKir2 subunits in Xenopus oocytes showed inwardly rectifying currents. The Ba2+ concentrations required for half-maximum block at -100 mV were 3.24 M for gpKir2.1, 0.51 M for gpKir2.2, 10.26 M for gpKir2.3 and 235 M for gpKir2.4. Ba2+ block of inward rectifier channels of cardiomyocytes was studied in cell-attached recordings. The concentration and voltage dependence of Ba2+ block of the large-conductance inward rectifier channels was virtually identical to that of gpKir2.2 expressed in Xenopus oocytes. Our results suggest that the large-conductance inward rectifier channels found in guinea-pig cardiomyocytes (34.0 pS) correspond to gpKir2.2. The intermediate-conductance (23.8 pS) and low-conductance (10.7 pS) channels described here may correspond to gpKir2.1 and gpKir2.3, respectively.
本研究的目的是比较克隆的Kir2通道与豚鼠心肌中天然整流通道的特性。通过筛选豚鼠心室的cDNA文库获得了gpKir2.1、gpKir2.2、gpKir2.3和gpKir2.4的cDNA。通过将cDNA与来自豚鼠基因组文库的核苷酸序列进行比较，推导了所有gpKir2基因的部分基因组结构。使用多细胞RT-PCR方法在分离的心肌细胞中研究了Kir2通道亚基的细胞特异性表达。发现gpKir2.1、gpKir2.2和gpKir2.3在心肌细胞中表达，而gpKir2.4不表达。用多克隆抗体进行的免疫细胞化学分析表明，Kir2.4的表达仅限于心脏中的神经细胞。在人胚肾细胞（HEK293）中进行转染后，发现对于gpKir2.1，对称K+条件下的平均单通道电导为30.6 pS；对于gpKir2.2为40.0 pS；对于Kir2.3为14.2 pS。在分离的豚鼠心肌细胞（n = 351）中进行的细胞贴附测量揭示了三个内向整流K+通道群体，其平均电导分别为34.0、23.8和10.7 pS。gpKir2亚基在非洲爪蟾卵母细胞中的表达显示出内向整流电流。在-100 mV时，使电流减半所需的Ba2+浓度对于gpKir2.1为3.24 μM，对于gpKir2.2为0.51 μM，对于gpKir2.3为10.26 μM , 对于gpKir2.4为235 μM。在细胞贴附记录中研究了心肌细胞内向整流通道的Ba2+阻断作用。大电导内向整流通道的Ba2+阻断浓度和电压依赖性与在非洲爪蟾卵母细胞中表达的gpKir2.2几乎相同。我们的结果表明，在豚鼠心肌细胞中发现的大电导内向整流通道（34.0 pS）对应于gpKir2.2。此处描述的中电导（23.8 pS）和低电导（10.7 pS）通道可能分别对应于gpKir2.1和gpKir2.3。
