|Coordinate||79,733,773 bp (GRCm38)|
|Base Change||G ⇒ T (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||Glutamic Acid changed to Stop codon|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000020581] [ENSMUSP00000097113]|
AA Change: E536*
|Predicted Effect||probably null|
AA Change: E536*
|Predicted Effect||probably null|
|Meta Mutation Damage Score||0.664|
|Is this an essential gene?||Non Essential (E-score: 0.000)|
|Candidate Explorer Status||CE: excellent candidate; human score: 2.5; ML prob: 0.85|
Linkage Analysis Data
|Alleles Listed at MGI|
|Mode of Inheritance||Unknown|
|Last Updated||2019-09-04 9:33 PM by Diantha La Vine|
|Record Created||2018-11-26 8:01 PM by Emre Turer|
The curveball2 phenotype was identified among G3 mice of the pedigree R6457, some of which showed reduced body weights compared to wild-type littermates (Figure 1). The mice also showed aberrant motor coordination as assessed by the rotarod test (Figure 2).
|Nature of Mutation|
Whole exome HiSeq sequencing of the G1 grandsire identified 68 mutations. All of the above anomalies were linked by continuous variable mapping to mutations in two genes on chromosome 10: Hcn2 and Prdm4. The mutation in Hcn2 was presumed causative as the phenotypes in the curveball2 mimicked that of other Hcn2 mutant mouse models [see curveball and (1;2)]. The mutation in Hcn2 is a G to T transversion at base pair 79,733,773 (v38) on chromosome 10, or base pair 17,140 in the GenBank genomic region NC_000076 encoding Hcn2. The strongest association was found with a recessive model of inheritance to the body weight phenotype, wherein one variant homozygote departed phenotypically from 10 homozygous reference mice and 16 heterozygous mice with a P value of 7.949 x 10-11 (Figure 3).
The mutation corresponds to residue 1,641 in the mRNA sequence NM_008226 within exon 6 of 8 total exons.
The mutated nucleotide is indicated in red. The mutation results in substitution of glutamic acid 536 for a premature stop codon (E536*) in the HCN2 protein.
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 (3). The HCN channels are complexes of four HCN subunits arranged around the central pore; the HCN proteins can form homomeric or heteromeric channels with other members of the HCN family (4-6).
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 (e.g., cyclic adenosine monophosphate [cAMP]) (7).
The curveball2 mutation results in substitution of glutamic acid 536 for a premature stop codon (E536*); Glu536 is within the cytoplasmic C-terminal tail preceding the CNBD.
Please see the record curveball for more information about Hcn2.
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 a 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 (8).
Loss-of-function mutations in HCN2 have been linked to idiopathic generalized epilepsies in patients (9;10). In addition, gain-of-function mutations are linked to polygenic epilepsy (11). Mutations in HCN2 have been correlated with increased incidence of febrile seizures (12). The mutant HCN2 channels exhibited faster kinetics with higher temperatures and subsequent increased rate of availability of the current.
Hcn2-deficient (Hcn2-/-) mice were hypoactive and smaller than their wild-type littermates (1;2;13). The Hcn2-/- mice exhibited absence epilepsy, ataxia, and sinus arrhythmia (13). Hcn2-/- mice also showed impaired balance and coordination (1;2).
The reduced size and the impaired coordination phenotypes observed in the curveball2 mice indicates a loss of HCN2-associated function.
curveball2(F):5'- AAAGTTCATCTGGTCCTGCC -3'
curveball2(R):5'- ATCCAGATCTCAGCCTTGCC -3'
curveball2_seq(F):5'- TGAGGGTTCACCAAGTAGCC -3'
curveball2_seq(R):5'- AGATCTCAGCCTTGCCACAGG -3'
1) 94°C 2:00
The following sequence of 410 nucleotides is amplified (chromosome 10, + strand):
1 aaagttcatc tggtcctgcc tccagcgtcc gcctgggaag ttagataggg gctgagggtt
Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.
1. Lewis, A. S., Vaidya, S. P., Blaiss, C. A., Liu, Z., Stoub, T. R., Brager, D. H., Chen, X., Bender, R. A., Estep, C. M., Popov, A. B., Kang, C. E., Van Veldhoven, P. P., Bayliss, D. A., Nicholson, D. A., Powell, C. M., Johnston, D., and Chetkovich, D. M. (2011) Deletion of the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Auxiliary Subunit TRIP8b Impairs Hippocampal Ih Localization and Function and Promotes Antidepressant Behavior in Mice. J Neurosci. 31, 7424-7440.
2. 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.
3. Santoro, B., Grant, S. G., Bartsch, D., and Kandel, E. R. (1997) Interactive Cloning with the SH3 Domain of N-Src Identifies a New Brain Specific Ion Channel Protein, with Homology to Eag and Cyclic Nucleotide-Gated Channels. Proc Natl Acad Sci U S A. 94, 14815-14820.
4. 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.
5. Altomare, C., Terragni, B., Brioschi, C., Milanesi, R., Pagliuca, C., Viscomi, C., Moroni, A., Baruscotti, M., and DiFrancesco, D. (2003) Heteromeric HCN1-HCN4 Channels: A Comparison with Native Pacemaker Channels from the Rabbit Sinoatrial Node. J Physiol. 549, 347-359.
6. Ulens, C., and Tytgat, J. (2001) Functional Heteromerization of HCN1 and HCN2 Pacemaker Channels. J Biol Chem. 276, 6069-6072.
7. Wainger, B. J., DeGennaro, M., Santoro, B., Siegelbaum, S. A., and Tibbs, G. R. (2001) Molecular Mechanism of cAMP Modulation of HCN Pacemaker Channels. Nature. 411, 805-810.
8. 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.
9. 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.
10. 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.
11. 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.
12. Nakamura, Y., Shi, X., Numata, T., Mori, Y., Inoue, R., Lossin, C., Baram, T. Z., and Hirose, S. (2013) Novel HCN2 Mutation Contributes to Febrile Seizures by Shifting the Channel's Kinetics in a Temperature-Dependent Manner. PLoS One. 8, e80376.
13. 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.
|Science Writers||Anne Murray|
|Illustrators||Diantha La Vine|
|Authors||Sohini Mukherjee, Zhao Zhang, and Bruce Beutler|