Stieber Juliane, Stöckl Georg, Herrmann Stefan, Hassfurth Benjamin, Hofmann Franz
Institut für Pharmakologie und Toxikologie der Technischen Universität München, Biedersteiner Strasse 29, 80802 München, Germany.
J Biol Chem. 2005 Oct 14;280(41):34635-43. doi: 10.1074/jbc.M502508200. Epub 2005 Jul 25.
Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels underlie the inward pacemaker current, termed I(f)/I(h), in a variety of tissues. Many details are known for the HCN subtypes 1, 2, and 4. We now successfully cloned the cDNA for HCN3 from human brain and compared the electrophysiological properties of hHCN3 to the other three HCN subtypes. Overexpression of human HCN3 channels in HEK293 cells resulted in a functional channel protein. Similar to hHCN2 channels, hHCN3 channels are activated with a rather slow time constant of 1244 +/- 526 ms at -100 mV, which is a greater time constant than that of HCN1 but a smaller one than that of HCN4 channels. The membrane potential for half-maximal activation V((1/2)) was -77 +/- 5.4 mV, and the reversal potential E(rev) was -20.5 +/- 4 mV, resulting in a permeability ratio P(Na)/P(K) of 0.3. Like all other HCNs, hHCN3 was inhibited rapidly and reversibly by extracellular cesium and slowly and irreversibly by extracellular applied ZD7288. Surprisingly, the human HCN3 channel was not modulated by intracellular cAMP, a hallmark of the other known HCN channels. Sequence comparison revealed >80% homology of the transmembrane segments, the pore region, and the cyclic nucleotide binding domain of hHCN3 with the other HCN channels. The missing response to cAMP distinguishes human HCN3 from both the well cAMP responding HCN subtypes 2 and 4 and the weak responding subtype 1.
超极化激活的环核苷酸门控阳离子（HCN）通道是多种组织中内向起搏电流（称为I(f)/I(h)）的基础。对于HCN亚型1、2和4，人们已经了解了许多细节。我们现在成功地从人脑中克隆了HCN3的cDNA，并将hHCN3的电生理特性与其他三种HCN亚型进行了比较。人HCN3通道在HEK293细胞中的过表达产生了一种功能性通道蛋白。与hHCN2通道类似，hHCN3通道在-100 mV时以相当慢的时间常数1244±526 ms被激活，该时间常数比HCN1的大，但比HCN4通道的小。半数最大激活的膜电位V((1/2))为-77±5.4 mV，反转电位E(rev)为-20.5±4 mV，导致通透性比P(Na)/P(K)为0.3。与所有其他HCN一样，hHCN3被细胞外铯快速可逆地抑制，并被细胞外应用的ZD7288缓慢不可逆地抑制。令人惊讶的是，人HCN3通道不受细胞内cAMP的调节，而细胞内cAMP是其他已知HCN通道的一个标志。序列比较显示，hHCN3的跨膜片段、孔区域和环核苷酸结合结构域与其他HCN通道具有>80%的同源性。对cAMP缺乏反应将人HCN3与cAMP反应良好的HCN亚型2和4以及反应较弱的亚型1区分开来。
