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Front Mol Neurosci
2015 Nov 03;8:63. doi: 10.3389/fnmol.2015.00063.
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An N-terminal deletion variant of HCN1 in the epileptic WAG/Rij strain modulates HCN current densities.
Wemhöner K
,
Kanyshkova T
,
Silbernagel N
,
Fernandez-Orth J
,
Bittner S
,
Kiper AK
,
Rinné S
,
Netter MF
,
Meuth SG
,
Budde T
,
Decher N
.
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Rats of the Wistar Albino Glaxo/Rij (WAG/Rij) strain show symptoms resembling human absence epilepsy. Thalamocortical neurons of WAG/Rij rats are characterized by an increased HCN1 expression, a negative shift in I h activation curve, and an altered responsiveness of I h to cAMP. We cloned HCN1 channels from rat thalamic cDNA libraries of the WAG/Rij strain and found an N-terminal deletion of 37 amino acids. In addition, WAG-HCN1 has a stretch of six amino acids, directly following the deletion, where the wild-type sequence (GNSVCF) is changed to a polyserine motif. These alterations were found solely in thalamus mRNA but not in genomic DNA. The truncated WAG-HCN1 was detected late postnatal in WAG/Rij rats and was not passed on to rats obtained from pairing WAG/Rij and non-epileptic August Copenhagen Irish rats. Heterologous expression in Xenopus oocytes revealed 2.2-fold increased current amplitude of WAG-HCN1 compared to rat HCN1. While WAG-HCN1 channels did not have altered current kinetics or changed regulation by protein kinases, fluorescence imaging revealed a faster and more pronounced surface expression of WAG-HCN1. Using co-expression experiments, we found that WAG-HCN1 channels suppress heteromeric HCN2 and HCN4 currents. Moreover, heteromeric channels of WAG-HCN1 with HCN2 have a reduced cAMP sensitivity. Functional studies revealed that the gain-of-function of WAG-HCN1 is not caused by the N-terminal deletion alone, thus requiring a change of the N-terminal GNSVCF motif. Our findings may help to explain previous observations in neurons of the WAG/Rij strain and indicate that WAG-HCN1 may contribute to the genesis of absence seizures in WAG/Rij rats.
FIGURE 1. Distribution of WAG-HCN1 channel in rat brain tissues. (A) Alignment of the N-terminus of the mRNA sequences from rat HCN1 and WAG-HCN1. The WAG-HCN1 mRNA has a deletion of 111 nucleotides, and 10 nucleotides following the deletion are exchanged (gray letters and boxes). The sequences displayed are integral parts of exon 1 of rat HCN1. (B) Alignment of the genomic sequences corresponding to the N-terminal mRNA of rat HCN1 and WAG-HCN1 displayed in (A). No differences were found (n = 5). (C) Upper panel, RT-PCR detection of HCN1 and WAG-HCN1 in different rat brain tissues: dLGN, ventrobasal thalamic complex (VB), hippocampus (Hippo), and S1 region of SSC. An additional PCR experiment of the dLGN with an expanded view of the double bands is illustrated in the middle (box). Lower panel, comparison of PCR detection of HCN1 and WAG-HCN1 (same experiment as the expanded view) in the LGN of WAG/Rij versus ACI rat after 27, 29, and 31 cycles. (D) RT-PCR detection of HCN1 using dLGN cDNA obtained from breaded WAG/Rij (W) and ACI (A) rats ( WAG/Rij x ACI and ACI x WAG/Rij, correspondingly). (E) PCR analysis of HCN1 and WAG-HCN1 isoforms in rat genomic DNA of WAG/Rij, ACl and their offspring. (F,G) Semi-quantitative RT-PCR analysis of HCN1 and WAG-HCN1 in the dLGN regions of WAG/Rij rats of different ages (P7, P30, P90). (F) Gel images of RT-PCR at P7. The numbers of PCR cycles are indicated. (G) Analysis of the peak intensities for PCR signals were plotted against cycle numbers for HCN1 and WAG-HCN1 at P7, P30 and P90. (C–G) The number of independent animals was n = 4 for all conditions.
FIGURE 2. Electrophysiological characterization of the WAG-HCN1 channel. (A) Alignment of the first 60 amino acids of the N-terminus from rat HCN1 and WAG-HCN1. The WAG-HCN1 channels have a deletion of 37 amino acids and six amino acids directly following the deletion (GNSVCF) are exchanged to serine residues. (B) Representative measurements of HCN1 (top) and WAG-HCN1 (bottom) currents. Oocytes were held at -30 mV and voltage steps of 2 s ranging from -30 to -140 mV were applied, followed by a step to -130 mV to record tail currents, see the illustrated voltage protocol. (C) Comparison of current amplitude of HCN1 (black), WAG-HCN1 (gray) and heteromeric HCN1/WAG-HCN1 currents (light gray), analyzed at -130 mV. The number of experiments are indicated in the bar graphs. (D) Conductance-voltage relationships of HCN1 (black), WAG-HCN1 (gray) and heteromeric HCN1/WAG-HCN1 currents (light gray). (E) Slow and fast time constants of activation at three different potentials, analyzed for HCN1 (black) and WAG-HCN1 (gray) using a bi-exponential fit. Right panel illustrates the amplitude ratio of the fast component over the sum of the fast and slow component. The number of experiments are indicated in the bar graphs. (F) Slow and fast time constants of deactivation analyzed at +20 mV for HCN1 (black) and WAG-HCN1 (gray) using bi-exponential fits. Right panel illustrates the amplitude ratio of the fast component of deactivation over the sum of the fast and slow component. The number of experiments are indicated in the bar graphs. ∗∗∗p < 0.001.
FIGURE 3. Co-expression of HCN1 or WAG-HCN1 with other HCN family members. (A) Representative current traces of the co-expression of HCN1 or WAG-HCN1 with HCN2. (B) Co-expression with WAG-HCN1 causes a significant decrease in current amplitude analyzed at -130 mV. The number of experiments are indicated in the bar graphs. (C) Co-expression of WAG-HCN1 with HCN4 shows a significant decrease in current amplitude analyzed at -130 mV. The number of experiments are indicated in the bar graphs. (D) Relative current amplitudes (at +40 mV) for Kv1.1 after co-expression with HCN1 or WAG-HCN1 with HCN2. The number of experiments are indicated in the bar graphs. ∗∗∗p < 0.001.
FIGURE 4. Properties of heteromeric channels of WAG-HCN1 with HCN2. (A) Conductance-voltage relationship of rat HCN1 co-expressed with HCN2 stored in theophyllin-free solutions, recorded before (black squares) and after 5 min of application of 2 mM 8-Br-cAMP (open squares). (B) Conductance-voltage relationship of WAG-HCN1 co-expressed with HCN2 stored in theophyllin-free solutions, recorded before (gray circles) and after 5 min of application of 2 mM 8-Br-cAMP (open circles). (C) V1/2 of activation for heteromeric channels of rat HCN1 or WAG-HCN1 co-expressed with HCN2. The number of experiments for (A–C) are indicated in parenthesis within the panel (C). ∗p < 0.05.
FIGURE 5. Molecular correlate for the WAG-HCN1 gain-of-function. (A) Cartoon depicting the N-termini of rat HCN1 (left) and WAG-HCN1 (right). The N-terminus of rat HCN1 has a length of 135 amino acids. WAG-HCN1 has a deletion of 37 amino acids and a novel polyserine motif of six amino acids. (B) Measurement of current amplitude of HCN1 and WAG-HCN1 48 h after injection and incubation in ND96 storage solution, supplemented with different protein kinase inhibitors (1 μM bisindolylmaleimide (BIS), 1 μM H-89, 10 μM genistein, or 1 μM staurosporine). Currents were normalized to the HCN1 construct incubated in normal storage solution. The number of experiments are indicated in the bar graphs. (C) Comparison of WAG-HCN1 (left) with a mutated WAG-HCN1 (HCN1-Δ37-aa) where the polyserine motif has been mutated to the wild-type sequence GNSVCF (right). This construct harbors only the deletion and not the polyserine stretch. (D) Current amplitudes of HCN1, WAG-HCN1 and HCN1-Δ37-aa analyzed at -130 mV. HCN1-Δ37-aa has a current amplitude similar to that of rat HCN1 (0.88 ± 0.08). The number of experiments are indicated in the bar graphs. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.
Akiyama,
Genistein, a specific inhibitor of tyrosine-specific protein kinases.
1987, Pubmed
Akiyama,
Genistein, a specific inhibitor of tyrosine-specific protein kinases.
1987,
Pubmed
Biel,
Hyperpolarization-activated cation channels: from genes to function.
2009,
Pubmed
Blumenfeld,
Early treatment suppresses the development of spike-wave epilepsy in a rat model.
2008,
Pubmed
Budde,
Impaired regulation of thalamic pacemaker channels through an imbalance of subunit expression in absence epilepsy.
2005,
Pubmed
Cain,
Thalamocortical neurons display suppressed burst-firing due to an enhanced Ih current in a genetic model of absence epilepsy.
2015,
Pubmed
Chen,
Properties of hyperpolarization-activated pacemaker current defined by coassembly of HCN1 and HCN2 subunits and basal modulation by cyclic nucleotide.
2001,
Pubmed
,
Xenbase
Chijiwa,
Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells.
1990,
Pubmed
Chow,
Energetics of cyclic AMP binding to HCN channel C terminus reveal negative cooperativity.
2012,
Pubmed
Chung,
Absence epilepsy in apathetic, a spontaneous mutant mouse lacking the h channel subunit, HCN2.
2009,
Pubmed
Coenen,
Genetic animal models for absence epilepsy: a review of the WAG/Rij strain of rats.
2003,
Pubmed
Couldwell,
Protein kinase C inhibitors induce apoptosis in human malignant glioma cell lines.
1994,
Pubmed
Crunelli,
Childhood absence epilepsy: genes, channels, neurons and networks.
2002,
Pubmed
Davis,
Potent selective inhibitors of protein kinase C.
1989,
Pubmed
Di Pasquale,
Increased excitability and inward rectification in layer V cortical pyramidal neurons in the epileptic mutant mouse Stargazer.
1997,
Pubmed
DiFrancesco,
Dysfunctional HCN ion channels in neurological diseases.
2015,
Pubmed
Gravante,
Interaction of the pacemaker channel HCN1 with filamin A.
2004,
Pubmed
Huguenard,
Thalamic synchrony and dynamic regulation of global forebrain oscillations.
2007,
Pubmed
Kanyshkova,
Postnatal expression pattern of HCN channel isoforms in thalamic neurons: relationship to maturation of thalamocortical oscillations.
2009,
Pubmed
Kanyshkova,
Differential regulation of HCN channel isoform expression in thalamic neurons of epileptic and non-epileptic rat strains.
2012,
Pubmed
Kay,
The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains.
2000,
Pubmed
Kole,
Inherited cortical HCN1 channel loss amplifies dendritic calcium electrogenesis and burst firing in a rat absence epilepsy model.
2007,
Pubmed
Kuisle,
Functional stabilization of weakened thalamic pacemaker channel regulation in rat absence epilepsy.
2006,
Pubmed
Leresche,
From sleep spindles of natural sleep to spike and wave discharges of typical absence seizures: is the hypothesis still valid?
2012,
Pubmed
Lewis,
Alternatively spliced isoforms of TRIP8b differentially control h channel trafficking and function.
2009,
Pubmed
Lolicato,
Tetramerization dynamics of C-terminal domain underlies isoform-specific cAMP gating in hyperpolarization-activated cyclic nucleotide-gated channels.
2011,
Pubmed
,
Xenbase
Ludwig,
Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2.
2003,
Pubmed
Lüttjohann,
Dynamics of networks during absence seizure's on- and offset in rodents and man.
2015,
Pubmed
Much,
Role of subunit heteromerization and N-linked glycosylation in the formation of functional hyperpolarization-activated cyclic nucleotide-gated channels.
2003,
Pubmed
Pan,
An N-Terminal ER Export Signal Facilitates the Plasma Membrane Targeting of HCN1 Channels in Photoreceptors.
2015,
Pubmed
,
Xenbase
Pinault,
Cellular and network mechanisms of genetically-determined absence seizures.
2005,
Pubmed
Rüegg,
Staurosporine, K-252 and UCN-01: potent but nonspecific inhibitors of protein kinases.
1989,
Pubmed
Santoro,
TRIP8b splice variants form a family of auxiliary subunits that regulate gating and trafficking of HCN channels in the brain.
2009,
Pubmed
Santoro,
Regulation of HCN channel surface expression by a novel C-terminal protein-protein interaction.
2004,
Pubmed
,
Xenbase
Schridde,
Environmental manipulations early in development alter seizure activity, Ih and HCN1 protein expression later in life.
2006,
Pubmed
Schulze-Bahr,
Pacemaker channel dysfunction in a patient with sinus node disease.
2003,
Pubmed
Strauss,
An impaired neocortical Ih is associated with enhanced excitability and absence epilepsy.
2004,
Pubmed
Streit,
A specific two-pore domain potassium channel blocker defines the structure of the TASK-1 open pore.
2011,
Pubmed
,
Xenbase
Vemana,
Intracellular Mg2+ is a voltage-dependent pore blocker of HCN channels.
2008,
Pubmed
,
Xenbase
Wainger,
Molecular mechanism of cAMP modulation of HCN pacemaker channels.
2001,
Pubmed
Yue,
Staurosporine-induced apoptosis in cardiomyocytes: A potential role of caspase-3.
1998,
Pubmed
Zolles,
Association with the auxiliary subunit PEX5R/Trip8b controls responsiveness of HCN channels to cAMP and adrenergic stimulation.
2009,
Pubmed
,
Xenbase
van Luijtelaar,
Spike-wave discharges in WAG/Rij rats are preceded by delta and theta precursor activity in cortex and thalamus.
2011,
Pubmed
van Luijtelaar,
Global and focal aspects of absence epilepsy: the contribution of genetic models.
2006,
Pubmed
van Luijtelaar,
Progress and outlooks in a genetic absence epilepsy model (WAG/Rij).
2014,
Pubmed
van Luijtelaar,
On the origin and suddenness of absences in genetic absence models.
2011,
Pubmed
