Different fluorescent labels report distinct components of spHCN channel voltage sensor movement.

We used voltage clamp fluorometry to probe the movement of the S4 helix in the voltage-sensing domain of the sea urchin HCN channel (spHCN) expressed in Xenopus oocytes. We obtained markedly different fluorescence responses with either ALEXA-488 or MTS-TAMRA covalently linked to N-terminal Cys332 of the S4 helix. With hyperpolarizing steps, ALEXA-488 fluorescence increased rapidly, consistent with it reporting the initial inward movement of S4, as previously described. In contrast, MTS-TAMRA fluorescence increased more slowly and its early phase correlated with that of channel opening. Additionally, a slow fluorescence component that tracked the development of the mode shift, or channel hysteresis, could be resolved with both labels. We quantitated this component as an increased deactivation tail current delay with concomitantly longer activation periods and found it to depend strongly on the presence of K+ ions in the pore. Using collisional quenching experiments and structural predictions, we established that ALEXA-488 was more exposed to solvent than MTS-TAMRA. We propose that components of S4 movement during channel activation can be kinetically resolved using different fluorescent probes to reveal distinct biophysical properties. Our findings underscore the need to apply caution when interpreting voltage clamp fluorometry data and demonstrate the potential utility of different labels to interrogate distinct biophysical properties of voltage-gated membrane proteins.

Drosophila HCN mediates gustatory homeostasis by preserving sensillar transepithelial potential in sweet environments.

Establishing transepithelial ion disparities is crucial for sensory functions in animals. In insect sensory organs called sensilla, a transepithelial potential, known as the sensillum potential (SP), arises through active ion transport across accessory cells, sensitizing receptor neurons such as mechanoreceptors and chemoreceptors. Because multiple receptor neurons are often co-housed in a sensillum and share SP, niche-prevalent overstimulation of single sensory neurons can compromise neighboring receptors by depleting SP. However, how such potential depletion is prevented to maintain sensory homeostasis remains unknown. Here, we find that the Ih-encoded hyperpolarization-activated cyclic nucleotide-gated (HCN) channel bolsters the activity of bitter-sensing gustatory receptor neurons (bGRNs), albeit acting in sweet-sensing GRNs (sGRNs). For this task, HCN maintains SP despite prolonged sGRN stimulation induced by the diet mimicking their sweet feeding niche, such as overripe fruit. We present evidence that Ih-dependent demarcation of sGRN excitability is implemented to throttle SP consumption, which may have facilitated adaptation to a sweetness-dominated environment. Thus, HCN expressed in sGRNs serves as a key component of a simple yet versatile peripheral coding that regulates bitterness for optimal food intake in two contrasting ways: sweet-resilient preservation of bitter aversion and the previously reported sweet-dependent suppression of bitter taste.

Thyroid Hormone Receptors in Control of Heart Rate.

Thyroid hormone has profound effects on cardiovascular functions, including heart rate. These effects can be mediated directly, for example, by changing the expression of target genes in the heart through nuclear thyroid hormone receptors, or indirectly by altering the autonomic nervous systems output of the brain. The underlying molecular mechanisms as well as the cellular substrates, however, are far from being understood. In this review, we summarize the recent key findings on the individual contributions of the two thyroid hormone receptor isoforms on the regulation of heart rate, challenging the role of the pacemaker channel genes Hcn2 and Hcn4 as sole mediators of the hormone's effect. Furthermore, we discuss the possible actions of thyroid hormone on the autonomic nervous system affecting heart rate distribution, and highlight the possibility of permanent alterations in heart and brain by impaired thyroid hormone action during development as important factors to consider when analyzing or designing experiments.

Propofol rescues voltage-dependent gating of HCN1 channel epilepsy mutants.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels1 are essential for pacemaking activity and neural signalling2,3. Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain4 and epileptic seizures5. The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs. 7,8). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies.

Multiple Exposures to Sevoflurane General Anesthesia During Pregnancy Inhibit CaMKII/CREB Axis by Downregulating HCN2 to Induce an Autism-Like Phenotype in Offspring Mice.

The objective of this investigation was to examine the impact of multiple exposures to general anesthesia (GA) with sevoflurane on the offspring of pregnant mice, as well as to elucidate the underlying mechanism. Neurodevelopmental assessments, including various reflexes and behavioral tests, were conducted on the offspring in the GA group to evaluate neuronal cell development. Furthermore, neonatal mouse neuronal cells were isolated and transfected with a high-expression CREB vector (pcDNA3.1-CREB), followed by treatment with sevoflurane (0.72 mol/L), ZD7288 (50 µmol/L), and KN-62 (10 µmol/L), or a combination of these compounds. The expression of relevant genes was then analyzed using qRT-PCR and western blot techniques. In comparison to the sham group, neonatal mice in the GA group exhibited significantly prolonged latencies in surface righting reflex, geotaxis test, and air righting reflex. Furthermore, there was a notable deceleration in the development of body weight and tail in the GA group. These mice also displayed impairments in social ability, reduced reciprocal social interaction behaviors, diminished learning capacity, and heightened levels of anxious behaviors. Additionally, synaptic trigger malfunction was observed, along with decreased production of c-Fos and neurotrophic factors. Sevoflurane was found to notably decrease cellular c-Fos and neurotrophic factor production, as well as the expression of HCN2 and CaMKII/CREB-related proteins. The inhibitory effects of sevoflurane on HCN2 or CaMKII channels were similar to those observed with ZD7288 or KN-62 inhibition. However, overexpression of CREB mitigated the impact of sevoflurane on neuronal cells. Repetitive exposure to sevoflurane general anesthesia while pregnant suppresses the CaMKII/CREB pathway, leading to the development of autism-like characteristics in offspring mice through the reduction of HCN2 expression.

HCN channels in the lateral habenula regulate pain and comorbid depressive-like behaviors in mice.

AIMS: Comorbid anxiodepressive-like symptoms (CADS) in chronic pain are closely related to the overactivation of the lateral habenula (LHb). Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have been implicated to play a key role in regulating neuronal excitability. However, the role of HCN channels in the LHb during CADS has not yet been characterized. This study aimed to investigate the effect of HCN channels in the LHb on CADS during chronic pain. METHODS: After chronic neuropathic pain induction by spared nerve injury (SNI), mice underwent a sucrose preference test, forced swimming test, tail suspension test, open-field test, and elevated plus maze test to evaluate their anxiodepressive-like behaviors. Electrophysiological recordings, immunohistochemistry, Western blotting, pharmacological experiments, and virus knockdown strategies were used to investigate the underlying mechanisms. RESULTS: Evident anxiodepressive-like behaviors were observed 6w after the SNI surgery, accompanied by increased neuronal excitability, enhanced HCN channel function, and increased expression of HCN2 isoforms in the LHb. Either pharmacological inhibition or virus knockdown of HCN2 channels significantly reduced LHb neuronal excitability and ameliorated both pain and depressive-like behaviors. CONCLUSION: Our results indicated that the LHb neurons were hyperactive under CADS in chronic pain, and this hyperactivation possibly resulted from the enhanced function of HCN channels and up-regulation of HCN2 isoforms.

Hyperpolarization-activated currents drive neuronal activation sequences in sleep.

Sequential neuronal patterns are believed to support information processing in the cortex, yet their origin is still a matter of debate. We report that neuronal activity in the mouse postsubiculum (PoSub), where a majority of neurons are modulated by the animal's head direction, was sequentially activated along the dorsoventral axis during sleep at the transition from hyperpolarized "DOWN" to activated "UP" states, while representing a stable direction. Computational modeling suggested that these dynamics could be attributed to a spatial gradient of hyperpolarization-activated currents (Ih), which we confirmed in ex vivo slice experiments and corroborated in other cortical structures. These findings open up the possibility that varying amounts of Ih across cortical neurons could result in sequential neuronal patterns and that traveling activity upstream of the entorhinal-hippocampal circuit organizes large-scale neuronal activity supporting learning and memory during sleep.

Structural determinants of ivabradine block of the open pore of HCN4.

HCN1-4 channels are the molecular determinants of the If/Ih current that crucially regulates cardiac and neuronal cell excitability. HCN dysfunctions lead to sinoatrial block (HCN4), epilepsy (HCN1), and chronic pain (HCN2), widespread medical conditions awaiting subtype-specific treatments. Here, we address the problem by solving the cryo-EM structure of HCN4 in complex with ivabradine, to date the only HCN-specific drug on the market. Our data show ivabradine bound inside the open pore at 3 Å resolution. The structure unambiguously proves that Y507 and I511 on S6 are the molecular determinants of ivabradine binding to the inner cavity, while F510, pointing outside the pore, indirectly contributes to the block by controlling Y507. Cysteine 479, unique to the HCN selectivity filter (SF), accelerates the kinetics of block. Molecular dynamics simulations further reveal that ivabradine blocks the permeating ion inside the SF by electrostatic repulsion, a mechanism previously proposed for quaternary ammonium ions.

High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons.

Pacemaking activity in substantia nigra dopaminergic neurons is generated by the coordinated activity of a variety of distinct somatodendritic voltage- and calcium-gated ion channels. We investigated whether these functional interactions could arise from a common localization in macromolecular complexes where physical proximity would allow for efficient interaction and co-regulations. For that purpose, we immunopurified six ion channel proteins involved in substantia nigra neuron autonomous firing to identify their molecular interactions. The ion channels chosen as bait were Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 channel proteins, and the methods chosen to determine interactions were co-immunoprecipitation analyzed through immunoblot and mass spectrometry as well as proximity ligation assay. A macromolecular complex composed of Cav1.3, HCN, and SK3 channels was unraveled. In addition, novel potential interactions between SK3 channels and sclerosis tuberous complex (Tsc) proteins, inhibitors of mTOR, and between HCN4 channels and the pro-degenerative protein Sarm1 were uncovered. In order to demonstrate the presence of these molecular interactions in situ, we used proximity ligation assay (PLA) imaging on midbrain slices containing the substantia nigra, and we could ascertain the presence of these protein complexes specifically in substantia nigra dopaminergic neurons. Based on the complementary functional role of the ion channels in the macromolecular complex identified, these results suggest that such tight interactions could partly underly the robustness of pacemaking in dopaminergic neurons.

Anisotropic Network Analysis of Open/Closed HCN4 Channel Advocates Asymmetric Subunit Cooperativity in cAMP Modulation of Gating.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are opened in an allosteric manner by membrane hyperpolarization and cyclic nucleotides such as cAMP. Because of conflicting reports from experimental studies on whether cAMP binding to the four available binding sites in the channel tetramer operates cooperatively in gating, we employ here a computational approach as a promising route to examine ligand-induced conformational changes after binding to individual sites. By combining an elastic network model (ENM) with linear response theory (LRT) for modeling the apo-holo transition of the cyclic nucleotide-binding domain (CNBD) in HCN channels, we observe a distinct pattern of cooperativity matching the "positive-negative-positive" cooperativity reported from functional studies. This cooperativity pattern is highly conserved among HCN subtypes (HCN4, HCN1), but only to a lesser extent visible in structurally related channels, which are only gated by voltage (KAT1) or cyclic nucleotides (TAX4). This suggests an inherent cooperativity between subunits in HCN channels as part of a ligand-triggered gating mechanism in these channels.

Structural basis for hyperpolarization-dependent opening of human HCN1 channel.

Hyperpolarization and cyclic nucleotide (HCN) activated ion channels are critical for the automaticity of action potentials in pacemaking and rhythmic electrical circuits in the human body. Unlike most voltage-gated ion channels, the HCN and related plant ion channels activate upon membrane hyperpolarization. Although functional studies have identified residues in the interface between the voltage-sensing and pore domain as crucial for inverted electromechanical coupling, the structural mechanisms for this unusual voltage-dependence remain unclear. Here, we present cryo-electron microscopy structures of human HCN1 corresponding to Closed, Open, and a putative Intermediate state. Our structures reveal that the downward motion of the gating charges past the charge transfer center is accompanied by concomitant unwinding of the inner end of the S4 and S5 helices, disrupting the tight gating interface observed in the Closed state structure. This helix-coil transition at the intracellular gating interface accompanies a concerted iris-like dilation of the pore helices and underlies the reversed voltage dependence of HCN channels.

Pacemaker Channels and the Chronotropic Response in Health and Disease.

Loss or dysregulation of the normally precise control of heart rate via the autonomic nervous system plays a critical role during the development and progression of cardiovascular disease-including ischemic heart disease, heart failure, and arrhythmias. While the clinical significance of regulating changes in heart rate, known as the chronotropic effect, is undeniable, the mechanisms controlling these changes remain not fully understood. Heart rate acceleration and deceleration are mediated by increasing or decreasing the spontaneous firing rate of pacemaker cells in the sinoatrial node. During the transition from rest to activity, sympathetic neurons stimulate these cells by activating ß-adrenergic receptors and increasing intracellular cyclic adenosine monophosphate. The same signal transduction pathway is targeted by positive chronotropic drugs such as norepinephrine and dobutamine, which are used in the treatment of cardiogenic shock and severe heart failure. The cyclic adenosine monophosphate-sensitive hyperpolarization-activated current (If) in pacemaker cells is passed by hyperpolarization-activated cyclic nucleotide-gated cation channels and is critical for generating the autonomous heartbeat. In addition, this current has been suggested to play a central role in the chronotropic effect. Recent studies demonstrate that cyclic adenosine monophosphate-dependent regulation of HCN4 (hyperpolarization-activated cyclic nucleotide-gated cation channel isoform 4) acts to stabilize the heart rate, particularly during rapid rate transitions induced by the autonomic nervous system. The mechanism is based on creating a balance between firing and recently discovered nonfiring pacemaker cells in the sinoatrial node. In this way, hyperpolarization-activated cyclic nucleotide-gated cation channels may protect the heart from sinoatrial node dysfunction, secondary arrhythmia of the atria, and potentially fatal tachyarrhythmia of the ventricles. Here, we review the latest findings on sinoatrial node automaticity and discuss the physiological and pathophysiological role of HCN pacemaker channels in the chronotropic response and beyond.

TBX3 transfection and nodal signal pathway inhibition promote differentiation of adipose mesenchymal stem cell to cardiac pacemaker-like cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are known as one of the best candidate cells to produce cardiac pacemaker-like cells (CPLCs). Upregulation of TBX3 transcription factor and inhibition of the nodal signal pathway have a significant role in the formation of cardiac pacemaker cells such as sinoatrial and atrioventricular nodes, which initiate the heartbeat and control the rhythm of heart contractions. This study aimed to confirm the effects of transfection of TBX3 transcription factor and inhibition of the nodal signal pathway on differentiating adipose-derived MSCs (AD-MSCs) to CPLCs. AD-MSCs were characterized using flow cytometry and three-lineage differentiation staining. METHODS: The transfection of TBX3 plasmid was carried out using lipofectamine, and inhibition of the nodal signal pathway was done using the small-molecule SB431542. The morphology of the cells was observed using a light microscope. Pacemaker-specific markers, including TBX3, Cx30, HCN4, HCN1, HCN3, and KCNN4, were evaluated using the qRT-PCR method. For protein level, TBX3 and Cx30 were evaluated using ELISA and immunofluorescence staining. The electrophysiology of cells was evaluated using a patch clamp. RESULTS: The TBX3 expression in the TBX3, SM, and TBX + SM groups significantly higher (p < 0.05) compared to the control group and cardiomyocytes. The expression of Cx40 and Cx43 genes were lower in TBX3, SM, TBX + SM groups. In contrast, Cx30 gene showed higher expression in TBX3 group. The expression HCN1, HCN3, and HCN4 genes are higher in TBX3 group. CONCLUSION: The transfection of TBX3 and inhibition of the nodal signal pathway by small-molecule SB431542 enhanced differentiation of AD-MSCs to CPLCs.

Cryo-EM structure of human HCN3 channel and its regulation by cAMP.

HCN channels are important for regulating heart rhythm and nerve activity and have been studied as potential drug targets for treating depression, arrhythmia, nerve pain, and epilepsy. Despite possessing unique pharmacological properties, HCN channels share common characteristics in that they are activated by hyperpolarization and modulated by cAMP and other membrane lipids. However, the mechanisms of how these ligands bind and modulate HCN channels are unclear. In this study, we solved structures of full-length human HCN3 using cryo-EM and captured two different states, including a state without any ligand bound and a state with cAMP bound. Our structures reveal the novel binding sites for cholesteryl hemisuccinate in apo state and show how cholesteryl hemisuccinate and cAMP binding cause conformational changes in different states. These findings explain how these small modulators are sensed in mammals at the molecular level. The results of our study could help to design more potent and specific compounds to influence HCN channel activity and offer new therapeutic possibilities for diseases that lack effective treatment.

Two contrasting mediodorsal thalamic circuits target the mouse medial prefrontal cortex.

At the heart of the prefrontal network is the mediodorsal (MD) thalamus. Despite the importance of MD in a broad range of behaviors and neuropsychiatric disorders, little is known about the physiology of neurons in MD. We injected the retrograde tracer cholera toxin subunit B (CTB) into the medial prefrontal cortex (mPFC) of adult wild-type mice. We prepared acute brain slices and used current clamp electrophysiology to measure and compare the intrinsic properties of the neurons in MD that project to mPFC (MD→mPFC neurons). We show that MD→mPFC neurons are located predominantly in the medial (MD-M) and lateral (MD-L) subnuclei of MD. MD-L→mPFC neurons had shorter membrane time constants and lower membrane resistance than MD-M→mPFC neurons. Relatively increased hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity in MD-L neurons accounted for the difference in membrane resistance. MD-L neurons had a higher rheobase that resulted in less readily generated action potentials compared with MD-M→mPFC neurons. In both cell types, HCN channels supported generation of burst spiking. Increased HCN channel activity in MD-L neurons results in larger after-hyperpolarization potentials compared with MD-M neurons. These data demonstrate that the two populations of MD→mPFC neurons have divergent physiologies and support a differential role in thalamocortical information processing and potentially behavior.NEW & NOTEWORTHY To realize the potential of circuit-based therapies for psychiatric disorders that localize to the prefrontal network, we need to understand the properties of the populations of neurons that make up this network. The mediodorsal (MD) thalamus has garnered attention for its roles in executive functioning and social/emotional behaviors mediated, at least in part, by its projections to the medial prefrontal cortex (mPFC). Here, we identify and compare the physiology of the projection neurons in the two MD subnuclei that provide ascending inputs to mPFC in mice. Differences in intrinsic excitability between the two populations of neurons suggest that neuromodulation strategies targeting the prefrontal thalamocortical network will have differential effects on these two streams of thalamic input to mPFC.

LRMP inhibits cAMP potentiation of HCN4 channels by disrupting intramolecular signal transduction.

Lymphoid restricted membrane protein (LRMP) is a specific regulator of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4, but the interaction domains, mechanisms of action, and basis for isoform-specificity remain unknown. Here, we identify the domains of LRMP essential for this regulation, show that LRMP acts by disrupting the intramolecular signal transduction between cyclic nucleotide binding and gating, and demonstrate that multiple unique regions in HCN4 are required for LRMP isoform-specificity. Using patch clamp electrophysiology and Förster resonance energy transfer (FRET), we identified the initial 227 residues of LRMP and the N-terminus of HCN4 as necessary for LRMP to associate with HCN4. We found that the HCN4 N-terminus and HCN4-specific residues in the C-linker are necessary for regulation of HCN4 by LRMP. Finally, we demonstrated that LRMP-regulation can be conferred to HCN2 by addition of the HCN4 N-terminus along with mutation of five residues in the S5 region and C-linker to the cognate HCN4 residues. Taken together, these results suggest that LRMP inhibits HCN4 through an isoform-specific interaction involving the N-terminals of both proteins that prevents the transduction of cAMP binding into a change in channel gating, most likely via an HCN4-specific orientation of the N-terminus, C-linker, and S4-S5 linker.

Ivabradine restores tonic cardiovascular autonomic control and reduces tachycardia, hypertension and left ventricular inflammation in post-weaning protein malnourished rats.

Malnutrition results in autonomic imbalance and heart hypertrophy. Overexpression of hyperpolarization-activated cyclic nucleotide-gated channels (HCN) in the left ventricles (LV) is linked to hypertrophied hearts and abnormal myocardium automaticity. Given that ivabradine (IVA) has emerging pleiotropic effects, in addition to the widely known bradycardic response, this study evaluated if IVA treatment could repair the autonomic control and cardiac damages in malnourished rats. AIM: Assess the impact of IVA on tonic cardiovascular autonomic control and its relationship with hemodynamics regulation, LV inflammation, and HCN gene expression in post-weaning protein malnutrition condition. MAIN METHODS: After weaning, male rats were divided into control (CG; 22 % protein) and malnourished (MG; 6 % protein) groups. At 35 days, groups were subdivided into CG-PBS, CG-IVA, MG-PBS and MG-IVA (PBS 1 ml/kg or IVA 1 mg/kg) received during 8 days. We performed jugular vein cannulation and electrode implant for drug delivery and ECG registration to assess tonic cardiovascular autonomic control; femoral cannulation for blood pressure (BP) and heart rate (HR) assessment; and LV collection to evaluate ventricular remodeling and HCN gene expression investigation. KEY FINDINGS: Malnutrition induced BP and HR increases, sympathetic system dominance, and LV remodeling without affecting HCN gene expression. IVA reversed the cardiovascular autonomic imbalance; prevented hypertension and tachycardia; and inhibited the LV inflammatory process and fiber thickening caused by malnutrition. SIGNIFICANCE: Our findings suggest that ivabradine protects against malnutrition-mediated cardiovascular damage. Moreover, our results propose these effects were not attributed to HCN expression changes, but rather to IVA pleiotropic effects on autonomic control and inflammation.

Cannabidiol potentiates hyperpolarization-activated cyclic nucleotide-gated (HCN4) channels.

Cannabidiol (CBD), the main non-psychotropic phytocannabinoid produced by the Cannabis sativa plant, blocks a variety of cardiac ion channels. We aimed to identify whether CBD regulated the cardiac pacemaker channel or the hyperpolarization-activated cyclic nucleotide-gated channel (HCN4). HCN4 channels are important for the generation of the action potential in the sinoatrial node of the heart and increased heart rate in response to ß-adrenergic stimulation. HCN4 channels were expressed in HEK 293T cells, and the effect of CBD application was examined using a whole-cell patch clamp. We found that CBD depolarized the V1/2 of activation in holo-HCN4 channels, with an EC50 of 1.6 µM, without changing the current density. CBD also sped activation kinetics by approximately threefold. CBD potentiation of HCN4 channels occurred via binding to the closed state of the channel. We found that CBD's mechanism of action was distinct from cAMP, as CBD also potentiated apo-HCN4 channels. The addition of an exogenous PIP2 analog did not alter the ability of CBD to potentiate HCN4 channels, suggesting that CBD also acts using a unique mechanism from the known HCN4 potentiator PIP2. Lastly, to gain insight into CBD's mechanism of action, computational modeling and targeted mutagenesis were used to predict that CBD binds to a lipid-binding pocket at the C-terminus of the voltage sensor. CBD represents the first FDA-approved drug to potentiate HCN4 channels, and our findings suggest a novel starting point for drug development targeting HCN4 channels.

Restoring thalamocortical circuit dysfunction by correcting HCN channelopathy in Shank3 mutant mice.

Thalamocortical (TC) circuits are essential for sensory information processing. Clinical and preclinical studies of autism spectrum disorders (ASDs) have highlighted abnormal thalamic development and TC circuit dysfunction. However, mechanistic understanding of how TC dysfunction contributes to behavioral abnormalities in ASDs is limited. Here, our study on a Shank3 mouse model of ASD reveals TC neuron hyperexcitability with excessive burst firing and a temporal mismatch relationship with slow cortical rhythms during sleep. These TC electrophysiological alterations and the consequent sensory hypersensitivity and sleep fragmentation in Shank3 mutant mice are causally linked to HCN2 channelopathy. Restoring HCN2 function early in postnatal development via a viral approach or lamotrigine (LTG) ameliorates sensory and sleep problems. A retrospective case series also supports beneficial effects of LTG treatment on sensory behavior in ASD patients. Our study identifies a clinically relevant circuit mechanism and proposes a targeted molecular intervention for ASD-related behavioral impairments.