Src tyrosine kinase alters gating of hyperpolarization-activated HCN4 pacemaker channel through Tyr531

We recently discovered that the constitutively active Src tyrosine kinase can enhance hyperpolarization-activated, cyclic nucleotide-gated (HCN) 4 channel activity by binding to the channel protein. To investigate the mechanism of modulation by Src of HCN channels, we studied the effects of a selective inhibitor of Src tyrosine kinase, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), on HCN4 and its mutant channels expressed in HEK 293 cells by using a whole cell patch-clamp technique. We found that PP2 can inhibit HCN4 currents by negatively shifting the voltage dependence of channel activation, decreasing the whole cell channel conductance, and slowing activation and deactivation kinetics. Screening putative tyrosine residues subject to phosphorylation yielded two candidates: Tyr(531) and Tyr(554). Substituting HCN4-Tyr(531) with phenylalanine largely abolished the effects of PP2 on HCN4 channels. Replacing HCN4-Tyr(554) with phenylalanine did not abolish the effects of PP2 on voltage-dependent activation but did eliminate PP2-induced slowing of channel kinetics. The inhibitory effects of HCN channels associated with reduced Src tyrosine activity is confirmed in HL-1 cardiomyocytes. Finally, we found that PP2 can decrease the heart rate in a mouse model. These results demonstrate that Src tyrosine kinase enhances HCN4 currents by shifting their activation to more positive potentials and increasing the whole cell channel conductance as well as speeding the channel kinetics. The tyrosine residue that mediates most of Src's actions on HCN4 channels is Tyr(531).     

S4 movement in a mammalian HCN channel

Hyperpolarization-activated, cyclic nucleotide-gated ion channels (HCN) mediate an inward cation current that contributes to spontaneous rhythmic firing activity in the heart and the brain. HCN channels share sequence homology with depolarization-activated Kv channels, including six transmembrane domains and a positively charged S4 segment. S4 has been shown to function as the voltage sensor and to undergo a voltage-dependent movement in the Shaker K+ channel (a Kv channel) and in the spHCN channel (an HCN channel from sea urchin). However, it is still unknown whether S4 undergoes a similar movement in mammalian HCN channels. In this study, we used cysteine accessibility to determine whether there is voltage-dependent S4 movement in a mammalian HCN1 channel. Six cysteine mutations (R247C, T249C, I251C, S253C, L254C, and S261C) were used to assess S4 movement of the heterologously expressed HCN1 channel in Xenopus oocytes. We found a state-dependent accessibility for four S4 residues: T249C and S253C from the extracellular solution, and L254C and S261C from the internal solution. We conclude that S4 moves in a voltage-dependent manner in HCN1 channels, similar to its movement in the spHCN channel. This S4 movement suggests that the role of S4 as a voltage sensor is conserved in HCN channels. In addition, to determine the reason for the different cAMP modulation and the different voltage range of activation in spHCN channels compared with HCN1 channels, we constructed a COOH-terminal-deleted spHCN. This channel appeared to be similar to a COOH-terminal-deleted HCN1 channel, suggesting that the main functional differences between spHCN and HCN1 channels are due to differences in their COOH termini or in the interaction between the COOH terminus and the rest of the channel protein in spHCN channels compared with HCN1 channels.     

超极化激活的环核苷酸门控通道（HCN 通道）抑制剂的研究进展

超极化激活的环核苷酸门控通道(HCN通道)有四个亚型,分别为HCN1-4。HCN通道各亚型之间的基本结构相似,在许多组织中均有表达,其中以大脑和心脏组织中表达最为丰富。HCN通道既参与所在组织的正常生理功能,也与所在组织的病理状态密切相关。如神经损伤引起的神经源性疼痛常检测到HCN1通道表达量的增加,肥厚性心肌病和终末期心力衰竭等病理状态下常检测到心室肌细胞HCN4 mRNA及HCN2 mRNA表达增加。鉴于HCN通道与许多疾病密切相关,因此,以其为靶点来治疗相关疾病成为可能,但是由于HCN通道分布广泛,而目前该通道阻滞剂均为非选择性亚型抑制剂,临床应用时不可避免的引起副反应,因此发展选择性HCN通道亚型抑制剂就显得刻不容缓。本文就HCN通道抑制剂的研究发展做进一步探讨。     

A novel mechanism of modulation of hyperpolarization-activated cyclic nucleotide-gated channels by Src kinase

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) play a crucial role in the regulation of cell excitability. Importantly, they contribute to spontaneous rhythmic activity in brain and heart. HCN channels are principally activated by membrane hyperpolarization and binding of cAMP. Here, we identify tyrosine phosphorylation by Src kinase as another mechanism affecting channel gating. Inhibition of Src by specific blockers slowed down activation kinetics of native and heterologously expressed HCN channels. The same effect on HCN channel activation was observed in cells cotransfected with a dominant-negative Src mutant. Immunoprecipitation demonstrated that Src binds to and phosphorylates native and heterologously expressed HCN2. Src interacts via its SH3 domain with a sequence of HCN2 encompassing part of the C-linker and the cyclic nucleotide binding domain. We identified a highly conserved tyrosine residue in the C-linker of HCN channels (Tyr476 in HCN2) that confers modulation by Src. Replacement of this tyrosine by phenylalanine in HCN2 or HCN4 abolished sensitivity to Src inhibitors. Mass spectrometry confirmed that Tyr476 is phosphorylated by Src. Our results have functional implications for HCN channel gating. Furthermore, they indicate that tyrosine phosphorylation contributes in vivo to the fine tuning of HCN channel activity.     

Two pacemaker channels from human heart with profoundly different activation kinetics

Cardiac pacemaking is produced by the slow diastolic depolarization phase of the action potential. The hyperpolarization-activated cation current (If) forms an important part of the pacemaker depolarization and consists of two kinetic components (fast and slow). Recently, three full-length cDNAs encoding hyperpolarization-activated and cyclic nucleotide-gated cation channels (HCN1-3) have been cloned from mouse brain. To elucidate the molecular identity of cardiac pacemaker channels, we screened a human heart cDNA library using a highly conserved neuronal HCN channel segment and identified two cDNAs encoding HCN channels. The hHCN2 cDNA codes for a protein of 889 amino acids. The HCN2 gene is localized on human chromosome 19p13.3 and contains eight exons spanning approximately 27 kb. The second cDNA, designated hHCN4, codes for a protein of 1203 amino acids. Northern blot and PCR analyses showed that both hHCN2 and hHCN4 are expressed in heart ventricle and atrium. When expressed in HEK 293 cells, either cDNA gives rise to hyperpolarization-activated cation currents with the hallmark features of native If. hHCN2 and hHCN4 currents differ profoundly from each other in their activation kinetics, being fast and slow, respectively. We thus conclude that hHCN2 and hHCN4 may underlie the fast and slow component of cardiac If, respectively.     

Flavonoid Regulation of HCN2 Channels

The hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are pacemaker channels whose currents contribute to rhythmic activity in the heart and brain. HCN channels open in response to hyperpolarizing voltages, and the binding of cAMP to their cyclic nucleotide-binding domain (CNBD) facilitates channel opening. Here, we report that, like cAMP, the flavonoid fisetin potentiates HCN2 channel gating. Fisetin sped HCN2 activation and shifted the conductance-voltage relationship to more depolarizing potentials with a half-maximal effective concentration (EC₅₀) of 1.8 μm. When applied together, fisetin and cAMP regulated HCN2 gating in a nonadditive fashion. Fisetin did not potentiate HCN2 channels lacking their CNBD, and two independent fluorescence-based binding assays reported that fisetin bound to the purified CNBD. These data suggest that the CNBD mediates the fisetin potentiation of HCN2 channels. Moreover, binding assays suggest that fisetin and cAMP partially compete for binding to the CNBD. NMR experiments demonstrated that fisetin binds within the cAMP-binding pocket, interacting with some of the same residues as cAMP. Together, these data indicate that fisetin is a partial agonist for HCN2 channels.     

Properties of hyperpolarization-activated pacemaker current defined by coassembly of HCN1 and HCN2 subunits and basal modulation by cyclic nucleotide

Members of the HCN channel family generate hyperpolarization-activated cation currents (Ih) that are directly regulated by cAMP and contribute to pacemaker activity in heart and brain. The four HCN isoforms show distinct but overlapping patterns of expression in different tissues. Here, we report that HCN1 and HCN2, isoforms coexpressed in neocortex and hippocampus that differ markedly in their biophysical properties, coassemble to generate heteromultimeric channels with novel properties. When expressed in Xenopus oocytes, HCN1 channels activate 5-10-fold more rapidly than HCN2 channels. HCN1 channels also activate at voltages that are 10-20 mV more positive than those required to activate HCN2. In cell-free patches, the steady-state activation curve of HCN1 channels shows a minimal shift in response to cAMP (+4 mV), whereas that of HCN2 channels shows a pronounced shift (+17 mV). Coexpression of HCN1 and HCN2 yields Ih currents that activate with kinetics and a voltage dependence that tend to be intermediate between those of HCN1 and HCN2 homomers, although the coexpressed channels do show a relatively large shift by cAMP (+14 mV). Neither the kinetics, steady-state voltage dependence, nor cAMP dose-response curve for the coexpressed Ih can be reproduced by the linear sum of independent populations of HCN1 and HCN2 homomers. These results are most simply explained by the formation of heteromeric channels with novel properties. The properties of these heteromeric channels closely resemble the properties of I(h) in hippocampal CA1 pyramidal neurons, cells that coexpress HCN1 and HCN2. Finally, differences in Ih channel properties recorded in cell-free patches versus intact oocytes are shown to be due, in part, to modulation of Ih by basal levels of cAMP in intact cells.     

Interaction of the pacemaker channel HCN1 with filamin A

Pacemaker channels are encoded by the HCN gene family and are responsible for a variety of cellular functions including control of spontaneous activity in cardiac myocytes and control of excitability in different types of neurons. Some of these functions require specific membrane localization. Although several voltage-gated channels are known to interact with intracellular proteins exerting auxiliary functions, no cytoplasmic proteins have been found so far to modulate HCN channels. Through the use of a yeast two-hybrid technique, here we showed that filamin A interacts with HCN1, an HCN isoform widely expressed in the brain, but not with HCN2 or HCN4. Filamin A is a cytoplasmic scaffold protein with actin-binding domains whose main function is to link transmembrane proteins to the actin cytoskeleton. Using several HCN1 C-terminal constructs, we identified a filamin A-interacting region of 22 amino acids located downstream from the cyclic nucleotide-binding domain; this region is not conserved in HCN2, HCN3, or HCN4. We also verified by immunoprecipitation from bovine brain that the filamin A-HCN1 interaction is functional in vivo. In filamin A-expressing cells (filamin+), HCN1 (but not HCN4) channels were expressed in hot spots, whereas they were evenly distributed on the membrane of cells lacking filamin A (filamin-) indicating that interaction with filamin A affects membrane localization. Also, in filamin- cells the gating kinetics of HCN1 were strongly accelerated relative to filamin+ cells. The interaction with filamin A may contribute to localizing HCN1 channels to specific neuronal areas and to modulating channel activity.     

Functional roles of charged residues in the putative voltage sensor of the HCN2 pacemaker channel

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels contribute to pacemaking activity in specialized neurons and cardiac myocytes. HCN channels have a structure similar to voltage-gated K(+) channels but have a much larger putative S4 transmembrane domain and open in response to membrane hyperpolarization instead of depolarization. As an initial attempt to define the structural basis of HCN channel gating, we have characterized the functional roles of the charged residues in the S2, S3, and S4 transmembrane domains. The nine basic residues and a single Ser in S4 were mutated individually to Gln, and the function of mutant channels was analyzed in Xenopus oocytes using two-microelectrode voltage clamp techniques. Surface membrane expression of hemagglutinin-epitope-tagged channel proteins was examined by chemiluminescence. Our results suggest that 1) Lys-291, Arg-294, Arg-297, and Arg-300 contribute to the voltage dependence of gating but not to channel folding or trafficking to the surface membrane; 2) Lys-303 and Ser-306 are essential for gating, but not for channel folding/trafficking; 3) Arg-312 is important for folding but not gating; and 4) Arg-309, Arg-315, and Arg-318 are crucial for normal protein folding/trafficking and may charge-pair with Asp residues located in the S2 and S3 domains.     

Expression of the hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 during mouse heart development

HCN4 is a hyperpolarization-activated nucleotide-gated cation channel involved in the generation of the I(f) current that drives cardiac pacemaker activity. Previous studies have demonstrated that HCN4 is highly expressed in a restricted manner in adult sinoatrial (SA) node [Eur. J. Biochem. 268 (2001) 1646]. However, its developmental expression pattern is unknown. We have examined expression of HCN4 mRNA during mouse heart development. HCN4 mRNA was first detected in the cardiac crescent at embryonic day (ED) 7.5. At ED 8 it was symmetrically located in the most caudal portion of the heart tube, the sinus venosus where pacemaker activity has previously been reported [Am. J. Physiol. 212 (1967) 407]. With further development, HCN4 expression became asymmetrically distributed, occupying the dorsal wall of the right atria, and was progressively restricted to the junction of the right atrial appendage and the superior vena cava. The site of HCN4 expression in late embryonic heart coincided with the location of the SA node in postnatal and adult heart [Cardiovasc. Res. 52 (2001) 51]. Our results suggest that HCN4 may be a unique marker of the developing SA node.     

Hyperpolarization-activated and cyclic nucleotide-gated channels are differentially expressed in juxtaglomerular cells in the olfactory bulb of mice

In the olfactory bulb, input from olfactory receptor neurons is processed by neuronal networks before it is relayed to higher brain regions. In many neurons, hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels generate and control oscillations of the membrane potential. Oscillations also appear crucial for information processing in the olfactory bulb. Four channel isoforms exist (HCN1–HCN4) that can form homo- or heteromers. Here, we describe the expression pattern of HCN isoforms in the olfactory bulb of mice by using a novel and comprehensive set of antibodies against all four isoforms. HCN isoforms are abundantly expressed in the olfactory bulb. HCN channels can be detected in most cell populations identified by commonly used marker antibodies. The combination of staining with marker and HCN antibodies has revealed at least 17 different staining patterns in juxtaglomerular cells. Furthermore, HCN isoforms give rise to an unexpected wealth of co-expression patterns but are rarely expressed in the same combination and at the same level in two given cell populations. Therefore, heteromeric HCN channels may exist in several cell populations in vivo. Our results suggest that HCN channels play an important role in olfactory information processing. The staining patterns are consistent with the possibility that both homomeric and heteromeric HCN channels are involved in oscillations of the membrane potential of juxtaglomerular cells.     

Up-regulation of Hyperpolarization-activated Cyclic Nucleotide-gated Channel 3 (HCN3) by Specific Interaction with K+ Channel Tetramerization Domain-containing Protein 3 (KCTD3)

Most ion channels consist of the principal ion-permeating core subunit(s) and accessory proteins that are assembled with the channel core. The biological functions of the latter proteins are diverse and include the regulation of the biophysical properties of the ion channel, its connection to signaling pathways and the control of its cell surface expression. There is recent evidence that native hyperpolarization-activated cyclic nucleotide-gated channel complexes (HCN1–4) also contain accessory subunits, among which TRIP8b (tetratricopeptide repeat-containing Rab8b-interacting protein) has been most extensively studied. Here, we identify KCTD3, a so far uncharacterized member of the potassium channel tetramerization-domain containing (KCTD) protein family as an HCN3-interacting protein. KCTD3 is widely expressed in brain and some non-neuronal tissues and colocalizes with HCN3 in specific regions of the brain including hypothalamus. Within the HCN channel family, KCTD3 specifically binds to HCN3 and leads to a profound up-regulation of cell surface expression and current density of this channel. HCN3 can also functionally interact with TRIP8b; however, we found no evidence for channel complexes containing both TRIP8b and KCTD3. The C terminus of HCN3 is crucially required for functional interaction with KCTD3. Replacement of the cytosolic C terminus of HCN2 by the corresponding domain of HCN3 renders HCN2 sensitive to regulation by KCTD3. The C-terminal-half of KCTD3 is sufficient for binding to HCN3. However, the complete protein including the N-terminal tetramerization domain is needed for HCN3 current up-regulation. Together, our experiments indicate that KCTD3 is an accessory subunit of native HCN3 complexes.     

A single histidine residue determines the pH sensitivity of the pacemaker channel HCN2

Hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels control the rhythmic activity of heart and neuronal networks. The activation of these channels is regulated in a complex manner by hormones and neurotransmitters. In addition it was suggested that the channels may be controlled by the pH of the cytosol. Here we demonstrate that HCN2, a member of the HCN channel family, is directly modulated by the intracellular pH in the physiological range. Protons inhibit HCN2 channels by shifting the voltage dependence of channel activation to more negative voltages. By using site-directed mutagenesis, we have identified a single histidine residue (His-321) localized at the boundary between the voltage-sensing S4 helix and the cytoplasmic S4-S5 linker of the channel that is a major determinant of pH sensitivity. Replacement of His-321 by either arginine, glutamine, or glutamate results in channels that are no longer sensitive to shifts in intracellular pH. In contrast, cAMP-mediated modulation is completely intact in mutant channels indicating that His-321 is not involved in the molecular mechanism that controls modulation of HCN channel activity by cyclic nucleotides. Because His-321 is conserved in all four HCN channels known so far, regulation by intracellular pH is likely to constitute a general feature of both cardiac and neuronal pacemaker channels.     

Functional expression of the human HCN3 channel

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.     

Regulation of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) Channel Activity by cCMP

Activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated in vivo by direct binding of the second messenger cAMP. This process plays a fundamental role in the fine-tuning of HCN channel activity and is critical for the modulation of cardiac and neuronal rhythmicity. Here, we identify the pyrimidine cyclic nucleotide cCMP as another regulator of HCN channels. We demonstrate that cCMP shifts the activation curves of two members of the HCN channel family, HCN2 and HCN4, to more depolarized voltages. Moreover, cCMP speeds up activation and slows down deactivation kinetics of these channels. The two other members of the HCN channel family, HCN1 and HCN3, are not sensitive to cCMP. The modulatory effect of cCMP is reversible and requires the presence of a functional cyclic nucleotide-binding domain. We determined an EC(50) value of ～30 μm for cCMP compared with 1 μm for cAMP. Notably, cCMP is a partial agonist of HCN channels, displaying an efficacy of ～0.6. cCMP increases the frequency of pacemaker potentials from isolated sinoatrial pacemaker cells in the presence of endogenous cAMP concentrations. Electrophysiological recordings indicated that this increase is caused by a depolarizing shift in the activation curve of the native HCN current, which in turn leads to an enhancement of the slope of the diastolic depolarization of sinoatrial node cells. In conclusion, our findings establish cCMP as a gating regulator of HCN channels and indicate that this cyclic nucleotide has to be considered in HCN channel-regulated processes.