Abstract
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.
Highlights
The functional expression of HCN3 channels proved to be difficult
Stable expression of hHCN3 in HEK293 cells allowed a more detailed characterization of this HCN channel and comparison to the other three human HCN subtypes. hHCN3/hHCN4 chimeric channels contributed to the understanding of how cAMP modulates HCN channels
Acceleration of activation, calculated from the time constants of activation, is about 2.8-fold (Fig. 7C). This suggests that the cyclic nucleotide binding domain (CNBD) of hHCN3 is capable of binding cyclic nucleotides despite the lack of modulation of the wild type hHCN3 channel by cAMP/cGMP. cAMP modulation, is no longer observed with the mutant channel h4CNBD/Ch3, which has in addition to the CNBD the remarkable short C terminus of hHCN3
Summary
Cloning of cDNAs Coding for HCN Channels—Human HCN3 cDNA was cloned from whole human brain QUICK-Clone cDNA (Clontech) using an overlapping PCR strategy. The two fragments with 1.1 and 1.3 kilobases, respectively, were ligated via the PflMI restriction site and cloned into the pcDNA3 mammalian expression vector (Invitrogen). After 3 weeks in selection medium, resistant cell clones were picked and checked for RNA transcription by Northern blotting, protein expression by Western blotting, and the presence of HCN currents by patch clamp recordings. DNA fragments containing the desired sequences were generated in several PCR steps using the hHCN3 and hHCN4 wild type cDNAs as templates and cloned into the HindIII/XbaI sites of the pcDNA3 vector. Lysates from HEK293 cells (5–10 g of total protein per lane) were applied on a 7.5% SDS-PAGE gel, separated, blotted, and probed with the antibodies using a chemiluminescence detection system.
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