|Coordinate||79,724,786 bp (GRCm38)|
|Base Change||T ⇒ A (forward strand)|
|Gene Name||hyperpolarization-activated, cyclic nucleotide-gated K+ 2|
|Chromosomal Location||79,716,634-79,736,108 bp (+)|
FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] The protein encoded by this gene is a hyperpolarization-activated cation channel involved in the generation of native pacemaker activity in the heart and in the brain. The encoded protein is activated by cAMP and can produce a fast, large current. Defects in this gene were noted as a possible cause of some forms of epilepsy. [provided by RefSeq, Jan 2017]
PHENOTYPE: Mice homozygous for mutant alleles exhibit decreased body weight, behavioral/neurological abnormalities, and tremors or absence seizures. [provided by MGI curators]
|Amino Acid Change||Isoleucine changed to Asparagine|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000020581] [ENSMUSP00000097113]|
AA Change: I317N
|Predicted Effect||probably damaging
PolyPhen 2 Score 0.998 (Sensitivity: 0.27; Specificity: 0.99)
AA Change: I317N
|Predicted Effect||probably damaging
PolyPhen 2 Score 0.998 (Sensitivity: 0.27; Specificity: 0.99)
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Live Mice|
|Last Updated||2019-02-05 12:17 PM by Diantha La Vine|
|Record Created||2016-07-07 1:06 PM by Jamie Russell|
The curveball phenotype was identified among N-nitroso-N-ethylurea (ENU)-mutagenized G3 mice of the pedigree R4507, some of which showed ataxia (Figure 1) and reduced body weights compared to wild-type littermates (Figure 2). Some mice also showed an increase in the CD4 to CD8 T cell ratio (Figure 3) caused by an increase in the frequency of CD4+ T cells in CD3+ T cells (Figure 4) with a concomitant decrease in the frequency of CD8+ T cells in CD3+ T cells (Figure 5), all in the peripheral blood.
|Nature of Mutation|
Whole exome HiSeq sequencing of the G1 grandsire identified 39 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Hcn2: a T to A transversion at base pair 79,724,786 (v38) on chromosome 10, or base pair 8,153 in the GenBank genomic region NC_000076 encoding Hcn2. The strongest association was found with a recessive model of linkage to the ataxia phenotype, wherein eight variant homozygotes departed phenotypically from 11 homozygous reference mice and 19 heterozygous mice with a P value of 4.513 x 10-8 (Figure 6). A substantial semidominant effect was observed with the ataxia phenotype but the mutation is preponderantly recessive.
The mutation corresponds to residue 985 in the mRNA sequence NM_178666 within exon 2 of 8 total exons.
The mutated nucleotide is indicated in red. The mutation results in an isoleucine (I) to asparagine (N) substitution at position 317 (I317N) in the HCN2 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 0.998).
Hcn2 encodes hyperpolarization-activated cyclic nucleotide-gated (HCN) channel 2 (HCN2). The HCN channels (i.e., HCN1, HCN2, HCN3, and HCN4) are members of the voltage-gated potassium ion channel superfamily present mainly in neurons and heart cells (1). The HCN channels are complexes of four HCN subunits arranged around a central pore; the HCN proteins can form homomeric or heteromeric channels with other members of the HCN family (2-4). The HCN proteins share 80 to 90% sequence identity between transmembrane domain 1 and to the end of the cyclic nucleotide-binding domain (CNBD). Most differences between the HCN proteins are within the N- and C-termini.
HCN2 has six transmembrane domains and cytoplasmic N- and C-termini. Transmembrane domain 4 is a positively charged voltage sensor. The ion-conducting pore region is between transmembrane domains 5 and 6. The HCN2 C-terminus has two domains: a C-linker domain composed of six α-helices separated by short loops and the CNBD. The C-linker domain connects the CNBD to the transmembrane domains. The CNBD mediates modulation by cyclic nucleotides (5). Cyclic adenosine monophosphate (cAMP) is the physiological agonist of HCN2 (6;7), but 3′,5′-Cyclic guanosine monophosphate (cGMP) and 3′,5′-cyclic cytidine monophosphate (cCMP) also bind to the CNBD and regulate the channel (6; 8-10)). The CNBD has four α-helices (A, P, B, C) with a β-roll (eight anti-parallel β-strands) between the A- and B-helices (9). The β-roll comprises eight β-strands in a jelly-roll-like topology. Cyclic nucleotides are bound inside the jelly-roll and interact with the β-roll and the C-helix. cAMP shifts the voltage activation range of HCN2 in the positive direction (7;11;12). Binding of cyclic nucleotides to the CNBD of HCN2 results in conformational changes that stabilize its open state (5;7;12). The cyclic nucleotides bind HCN2 between the β-roll and the C-helix (6). Upon cAMP binding, the C-helix moves in toward the β-roll (9;10;13).The conformation of the CNBD after cAMP, cGMP, and cCMP binding are similar, but the equilibrium between conformations varies between the ligands, and ligand specificity differs from the intact channel.
HCN2 associates with several proteins to regulate its function. HCN2 is trafficked to dendrites through binding to the chaperone TRIP8b (tetratricopeptide repeat (TPR)-containing Rab8b interacting protein) (12;14-16). Reduced TRIP8b expression resulted in reduced HCN surface expression and disruption of the normal gradient of HCN subunits in CA1 pyramidal neurons (17). HCN2 forms a complex with the scaffold proteins tamalin, S-SCAM, and Mint2 (18). Assembly with the scaffold proteins promotes the correct distribution, trafficking, and clustering of ion channels. Tamalin interacts with the PDZ-binding motif (S/XS/ANL/M in the HCN proteins; SRLSSNL in HCN2) and the C-terminal tail of HCN2. S-SCAM binds at the CNBD and the sequence downstream of the CNBD in the C-terminal tail of HCN2. The sequence downstream of the HCN2 CNBD interacts with Mint2.
Several residues in HCN2 have specific functions. HCN2 is N-linked glycosylated at Asn380. N-linked glycosylation of the HCN channels is required for trafficking to the plasma membrane and for its stability (19). All four subunits of the tetrameric HCN2 channel do not have to be glycosylated for HCN2 channels to insert into cell membranes. His321 is a major determinant of pH sensitivity (20). His321 is at the boundary between transmembrane domain 4 and the cytoplasmic loop between transmembrane 4 and 5. Mutation of His321 to arginine, glutamine, or glutamate causes loss in intracellular pH sensitivity. Mutation of His321 does not alter cAMP-mediated modulation of the HCN2 channel. Lys291, Arg294, Arg297, and Arg300 contribute to the voltage dependence of gating, but are not required for trafficking or folding (21). Lys303 and Ser306 are required for gating, but are not required for trafficking or folding. Arg312 is required for folding, but not gating. Arg309, Arg315, and Arg318 are required for HCN2 folding and trafficking.
The curveball mutation results in an isoleucine (I) to asparagine (N) substitution at position 317 (I317N) within the cytoplasmic loop between transmembrane domains 4 and 5. Residue 317 is within the proximity of Arg315 and Arg318, which are required for HCN2 folding and trafficking.
HCN2 is ubiquitously expressed throughout the central nervous system, with highest expression in the thalamus and brain stem nuclei [(1;22-24); reviewed in (25)]. HCN2 is predominantly localized mainly in pyramidal cell dendrites. HCN2 is also found at lower concentrations in the somata of pyramidal neurons as well as other neuron subtypes [reviewed in (25)]. HCN2 is also expressed in the heart (26-28).
HCN2 is a regulator of nociceptor excitability. In nociceptive neurons, HCN2 modulates the generation of action potentials in response to inflammation. For example, upon prostaglandin E2 (PGE2) binding to a G protein-coupled receptor coupled to Gs, adenylate cyclase is activated, which elevates cAMP. The elevation of cAMP shifts the activation curve of HCN2 in the positive direction, which causes an HCN2-dependent tonic inward current (termed Ih) to be activated at the resting potential. The Ih current is essential for cardiac and neuronal pacemaker activity, dendritic integration of synaptic transmission, and the setting of resting potentials (18).
Hcn2-deficient (Hcn2-/-) mice were hypoactive and smaller than their wild-type littermates (29). The Hcn2-/- mice exhibited absence epilepsy, ataxia, and sinus arrhythmia (29). Mice with a cardiomyocyte-specific deletion of Hcn2 also exhibited sinus arrhythmia indicating that the heart phenotype is independent of the neuronal defects. Heart rate was not affected. Mice in which HCN2 is only deleted in the NaV1.8-expressing population of sensory neurons are normal (11). NaV1.8 is expressed only in nociceptive primary sensory neurons. These mice have an absence of hyperalgesia when tested with heat after injection with the inflammatory stimuli PGE2 or carrageenan. The mice also show an attenuated response in the late phase of the formalin model. They exhibit normal hyperalgesia to mechanical stimuli post-inflammation. Without inflammation, the mice exhibit normal withdrawal thresholds to acute noxious stimuli. Neuron-specific loss of HCN2 expression caused reduced tactile hypersensitivity after exposure to complete Freund's adjuvant (a chronic inflammatory pain model), but heat hypersensitivity was unaffected (30). The apathetic (ap/ap) mouse model has a spontaneous Hcn2 mutation that results in ataxia, absence seizures, and generalized tonic-clonic seizures (17). Heterozygous apathetic (ap/+) mice have a normal gait and occasional absence seizures.
Loss-of-function mutations in human HCN2 have been linked to idiopathic generalized epilepsies in patients (31;32). In addition, gain-of-function mutations are linked to polygenic epilepsy (33). Mutations in HCN2 have been correlated with increased incidence of febrile seizures (34). The mutant HCN2 channels exhibited faster kinetics with higher temperatures and subsequent increased rate of availability of the current.
The reduced size and the ataxia phenotype observed in the curveball mice indicates a loss of HCN2curveball function.
curveball(F):5'- GGACAACACGGAGATCATCC -3'
curveball(R):5'- TGCCCTGTCCACAATCAAG -3'
curveball_seq(F):5'- GATCATCCTGGACCCCGAGAAG -3'
curveball_seq(R):5'- TGTCCACAATCAAGGCCCC -3'
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2. Whitaker, G. M., Angoli, D., Nazzari, H., Shigemoto, R., and Accili, E. A. (2007) HCN2 and HCN4 Isoforms Self-Assemble and Co-Assemble with Equal Preference to Form Functional Pacemaker Channels. J Biol Chem. 282, 22900-22909.
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6. DeBerg, H. A., Brzovic, P. S., Flynn, G. E., Zagotta, W. N., and Stoll, S. (2016) Structure and Energetics of Allosteric Regulation of HCN2 Ion Channels by Cyclic Nucleotides. J Biol Chem. 291, 371-381.
7. Craven, K. B., and Zagotta, W. N. (2006) CNG and HCN Channels: Two Peas, One Pod. Annu Rev Physiol. 68, 375-401.
8. Zong, X., Krause, S., Chen, C. C., Kruger, J., Gruner, C., Cao-Ehlker, X., Fenske, S., Wahl-Schott, C., and Biel, M. (2012) Regulation of Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channel Activity by cCMP. J Biol Chem. 287, 26506-26512.
9. Zagotta, W. N., Olivier, N. B., Black, K. D., Young, E. C., Olson, R., and Gouaux, E. (2003) Structural Basis for Modulation and Agonist Specificity of HCN Pacemaker Channels. Nature. 425, 200-205.
10. Flynn, G. E., Black, K. D., Islas, L. D., Sankaran, B., and Zagotta, W. N. (2007) Structure and Rearrangements in the Carboxy-Terminal Region of SpIH Channels. Structure. 15, 671-682.
11. Emery, E. C., Young, G. T., Berrocoso, E. M., Chen, L., and McNaughton, P. A. (2011) HCN2 Ion Channels Play a Central Role in Inflammatory and Neuropathic Pain. Science. 333, 1462-1466.
12. Robinson, R. B., and Siegelbaum, S. A. (2003) Hyperpolarization-Activated Cation Currents: From Molecules to Physiological Function. Annu Rev Physiol. 65, 453-480.
13. Puljung, M. C., DeBerg, H. A., Zagotta, W. N., and Stoll, S. (2014) Double Electron-Electron Resonance Reveals cAMP-Induced Conformational Change in HCN Channels. Proc Natl Acad Sci U S A. 111, 9816-9821.
14. Biel, M., Wahl-Schott, C., Michalakis, S., and Zong, X. (2009) Hyperpolarization-Activated Cation Channels: From Genes to Function. Physiol Rev. 89, 847-885.
15. Wahl-Schott, C., and Biel, M. (2009) HCN Channels: Structure, Cellular Regulation and Physiological Function. Cell Mol Life Sci. 66, 470-494.
16. Shah, M. M., Hammond, R. S., and Hoffman, D. A. (2010) Dendritic Ion Channel Trafficking and Plasticity. Trends Neurosci. 33, 307-316.
17. Chung, W. K., Shin, M., Jaramillo, T. C., Leibel, R. L., LeDuc, C. A., Fischer, S. G., Tzilianos, E., Gheith, A. A., Lewis, A. S., and Chetkovich, D. M. (2009) Absence Epilepsy in Apathetic, a Spontaneous Mutant Mouse Lacking the h Channel Subunit, HCN2. Neurobiol Dis. 33, 499-508.
18. Kimura, K., Kitano, J., Nakajima, Y., and Nakanishi, S. (2004) Hyperpolarization-Activated, Cyclic Nucleotide-Gated HCN2 Cation Channel Forms a Protein Assembly with Multiple Neuronal Scaffold Proteins in Distinct Modes of Protein-Protein Interaction. Genes Cells. 9, 631-640.
19. Li, M., Tonggu, L., Tang, L., and Wang, L. (2015) Effects of N-Glycosylation on Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channels. Biochem J. 466, 77-84.
20. Zong, X., Stieber, J., Ludwig, A., Hofmann, F., and Biel, M. (2001) A Single Histidine Residue Determines the pH Sensitivity of the Pacemaker Channel HCN2. J Biol Chem. 276, 6313-6319.
21. Chen, J., Mitcheson, J. S., Lin, M., and Sanguinetti, M. C. (2000) Functional Roles of Charged Residues in the Putative Voltage Sensor of the HCN2 Pacemaker Channel. J Biol Chem. 275, 36465-36471.
22. Santoro, B., Chen, S., Luthi, A., Pavlidis, P., Shumyatsky, G. P., Tibbs, G. R., and Siegelbaum, S. A. (2000) Molecular and Functional Heterogeneity of Hyperpolarization-Activated Pacemaker Channels in the Mouse CNS. J Neurosci. 20, 5264-5275.
23. Notomi, T., and Shigemoto, R. (2004) Immunohistochemical Localization of Ih Channel Subunits, HCN1-4, in the Rat Brain. J Comp Neurol. 471, 241-276.
24. Moosmang, S., Biel, M., Hofmann, F., and Ludwig, A. (1999) Differential Distribution of Four Hyperpolarization-Activated Cation Channels in Mouse Brain. Biol Chem. 380, 975-980.
25. Shah, M. M. (2014) Cortical HCN Channels: Function, Trafficking and Plasticity. J Physiol. 592, 2711-2719.
26. Shi, W., Wymore, R., Yu, H., Wu, J., Wymore, R. T., Pan, Z., Robinson, R. B., Dixon, J. E., McKinnon, D., and Cohen, I. S. (1999) Distribution and Prevalence of Hyperpolarization-Activated Cation Channel (HCN) mRNA Expression in Cardiac Tissues. Circ Res. 85, e1-6.
27. Moosmang, S., Stieber, J., Zong, X., Biel, M., Hofmann, F., and Ludwig, A. (2001) Cellular Expression and Functional Characterization of Four Hyperpolarization-Activated Pacemaker Channels in Cardiac and Neuronal Tissues. Eur J Biochem. 268, 1646-1652.
28. Ludwig, A., Zong, X., Stieber, J., Hullin, R., Hofmann, F., and Biel, M. (1999) Two Pacemaker Channels from Human Heart with Profoundly Different Activation Kinetics. EMBO J. 18, 2323-2329.
29. Ludwig, A., Budde, T., Stieber, J., Moosmang, S., Wahl, C., Holthoff, K., Langebartels, A., Wotjak, C., Munsch, T., Zong, X., Feil, S., Feil, R., Lancel, M., Chien, K. R., Konnerth, A., Pape, H. C., Biel, M., and Hofmann, F. (2003) Absence Epilepsy and Sinus Dysrhythmia in Mice Lacking the Pacemaker Channel HCN2. EMBO J. 22, 216-224.
30. Schnorr, S., Eberhardt, M., Kistner, K., Rajab, H., Kasser, J., Hess, A., Reeh, P., Ludwig, A., and Herrmann, S. (2014) HCN2 Channels Account for Mechanical (but Not Heat) Hyperalgesia during Long-Standing Inflammation. Pain. 155, 1079-1090.
31. DiFrancesco, J. C., Barbuti, A., Milanesi, R., Coco, S., Bucchi, A., Bottelli, G., Ferrarese, C., Franceschetti, S., Terragni, B., Baruscotti, M., and DiFrancesco, D. (2011) Recessive Loss-of-Function Mutation in the Pacemaker HCN2 Channel Causing Increased Neuronal Excitability in a Patient with Idiopathic Generalized Epilepsy. J Neurosci. 31, 17327-17337.
32. Tang, B., Sander, T., Craven, K. B., Hempelmann, A., and Escayg, A. (2008) Mutation Analysis of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels HCN1 and HCN2 in Idiopathic Generalized Epilepsy. Neurobiol Dis. 29, 59-70.
33. Dibbens, L. M., Reid, C. A., Hodgson, B., Thomas, E. A., Phillips, A. M., Gazina, E., Cromer, B. A., Clarke, A. L., Baram, T. Z., Scheffer, I. E., Berkovic, S. F., and Petrou, S. (2010) Augmented Currents of an HCN2 Variant in Patients with Febrile Seizure Syndromes. Ann Neurol. 67, 542-546.
|Science Writers||Anne Murray|
|Authors||Jamie Russell, Emre Turer, Xue Zhong, and Bruce Beutler|