Abstract
Hyperpolarization-activated cAMP-regulated (HCN) channels play important physiological roles in both cardiovascular and central nervous systems. Among the four HCN isoforms, HCN2 and HCN4 show high expression levels in the human heart, with HCN4 being the major cardiac isoform. The previously published crystal structure of the mouse HCN2 (mHCN2) C-terminal fragment, including the C-linker and the cyclic-nucleotide binding domain (CNBD), has provided many insights into cAMP-dependent gating in HCN channels. However, structures of other mammalian HCN channel isoforms have been lacking. Here we used a combination of approaches including structural biology, biochemistry, and electrophysiology to study cAMP-dependent gating in HCN4 channel. First we solved the crystal structure of the C-terminal fragment of human HCN4 (hHCN4) channel at 2.4 Å. Overall we observed a high similarity between mHCN2 and hHCN4 crystal structures. Functional comparison between two isoforms revealed that compared with mHCN2, the hHCN4 protein exhibited marked different contributions to channel function, such as a ∼3-fold reduction in the response to cAMP. Guided by structural differences in the loop region between β4 and β5 strands, we identified residues that could partially account for the differences in response to cAMP between mHCN2 and hHCN4 proteins. Moreover, upon cAMP binding, the hHCN4 C-terminal protein exerts a much prolonged effect in channel deactivation that could have significant physiological contributions.
Highlights
37082 JOURNAL OF BIOLOGICAL CHEMISTRY gous to other cNMP-binding proteins downstream from the last transmembrane domain (S6) on the intracellular side [2,3,4,5]
Functional Differences between the mHCN2 and hHCN4 C-terminal Fragments—Prompted by the differential distribution of HCN2 and HCN4 isoforms in the heart and the different responses to cAMP, we carried out electrophysiological experiments to analyze the function of the human HCN4 C-terminal fragment and compare it to the corresponding part from mouse HCN2, which had been crystallized and well characterized [10]
The K1⁄2 value measured from this mHCN2-h4 chimera should be close to that of full-length hHCN4 channel, as it was previously shown that the response to cAMP was largely determined by the C terminus, especially the C-linker and cyclic nucleotide binding domain (CNBD) [33]
Summary
Functional Expression in Xenopus Oocytes and Electrophysiological Characterization—To make a chimera between hHCN4 and mHCN2, we first amplified by PCR the C-linker and CNBD region from hHCN4. The resulting plasmid, pSMT3-hHCN4C, was transformed into Escherichia coli BL21(DE3) Gold (Novagen) cells where hHCN4C was expressed as a Smt fusion protein with a N-terminal His tag. After cleaving the Smt tag with Ulp protease, the protein sample was dialyzed overnight in a buffer containing 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 1 mM DTT. The purified protein was concentrated to Ͼ10 mg/ml in buffer containing 5 mM MES-NaOH, pH 6.0, and 50 mM NaCl. The pSMT3 vector-Ulp protease system was a generous gift from Dr Chris Lima. Sedimentation Equilibrium—Concentrated protein samples were separated into two pools and dialyzed against buffers containing (in mM) 20 HEPES, 300 NaCl, and 1 DTT, pH 7.2, either with or without 5 mM cAMP. We used VMD [32] and PYMOL (The PyMOL Molecular Graphics System, Schrodinger, LLC.) for structure presentation
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