Benedetta Terragni

Heteromeric HCN1–HCN4 channels: a comparison with native pacemaker channels from the rabbit sinoatrial node

‘Funny‐’ (f‐) channels of cardiac sino‐atrial node (SAN) cells are key players in the process of pacemaker generation and mediate the modulatory action of autonomic transmitters on heart rate. The molecular components of f‐channels are the hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channels. Of the four HCN isoforms known, two (HCN4 and HCN1) are expressed in the rabbit SAN at significant levels. However, the properties of f‐channels of SAN cells do not conform to specific features of the two isoforms expressed locally. For example, activation kinetics and cAMP sensitivity of native pacemaker channels are intermediate between those reported for HCN1 and HCN4. Here we have explored the possibility that both HCN4 and HCN1 isoforms contribute to the native If in SAN cells by co‐assembling into heteromeric channels. To this end, we used heterologous expression in human embryonic kidney (HEK) 293 cells to investigate the kinetics and cAMP response of the current generated by co‐transfected (HCN4 + HCN1) and concatenated (HCN4‐HCN1 (4–1) tandem or HCN1‐HCN4 (1–4) tandem) rabbit constructs and compared them with those of the native f‐current from rabbit SAN. 4–1 tandem, but not co‐transfected, currents had activation kinetics approaching those of If; however, the activation range of 4–1 tandem channels was more negative than that of the f‐channel and their cAMP sensitivity were poorer (although that of 1–4 tandem channels was normal). Co‐transfection of 4–1 tandem channels with minK‐related protein 1(MiRP1) did not alter their properties. HCN1 and HCN4 may contribute to native f‐channels, but a ‘context’‐dependent mechanism is also likely to modulate the channel properties in native tissues.

Localization of Pacemaker Channels in Lipid Rafts Regulates Channel Kinetics

Lipid rafts are discrete membrane subdomains rich in sphingolipids and cholesterol. In ventricular myocytes a function of caveolae, a type of lipid rafts, is to concentrate in close proximity several proteins of the β-adrenergic transduction pathway. We have investigated the subcellular localization of HCN4 channels expressed in HEK cells and studied the effects of such localization on the properties of pacemaker channels in HEK and rabbit sinoatrial (SAN) cells. We used a discontinuous sucrose gradient and Western blot analysis to detect HCN4 proteins in HEK and in SAN cells, and found that HCN4 proteins localize to low-density membrane fractions together with flotillin (HEK) or caveolin-3 (SAN), structural proteins of caveolae. Lipid raft disruption by cell incubation with methyl-β-cyclodextrin (MβCD) impaired specific HCN4 localization. It also shifted the midpoint of activation of the HCN4 current in HEK cells and of If in SAN cells to the positive direction by 11.9 and 10.4 mV, respectively. These latter effects were not due to elevation of basal cyclic nucleotide levels because the cholesterol-depletion treatment did not alter the current response to cyclic nucleotides. In accordance with an increased If, MβCD-treated SAN cells showed large increases of diastolic depolarization slope (87%) and rate (58%). We also found that the kinetics of HCN4- and native f-channel deactivation were slower after lipid raft disorganization. In conclusion, our work indicates that pacemaker channels localize to lipid rafts and that disruption of lipid rafts causes channels to redistribute within the membrane and modifies their kinetic properties.

Self-Limited Hyperexcitability: Functional Effect of a Familial Hemiplegic Migraine Mutation of the Nav1.1 (SCN1A) Na+ Channel

Familial hemiplegic migraine (FHM) is an autosomal dominant inherited subtype of severe migraine with aura. Mutations causing FHM (type 3) have been identified in SCN1A, the gene encoding neuronal voltage-gated Nav1.1 Na+ channel α subunit, but functional studies have been done using the cardiac Nav1.5 isoform, and the observed effects were similar to those of some epileptogenic mutations. We studied the FHM mutation Q1489K by transfecting tsA-201 cells and cultured neurons with human Nav1.1. We show that the mutation has effects on the gating properties of the channel that can be consistent with both hyperexcitability and hypoexcitability. Simulation of neuronal firing and long depolarizing pulses mimicking promigraine conditions revealed that the effect of the mutation is a gain of function consistent with increased neuronal firing. However, during high-frequency discharges and long depolarizations, the effect became a loss of function. Recordings of firing of transfected neurons showed higher firing frequency at the beginning of long discharges. This self-limited capacity to induce neuronal hyperexcitability may be a specific characteristic of migraine mutations, able to both trigger the cascade of events that leads to migraine and counteract the development of extreme hyperexcitability typical of epileptic seizures. Thus, we found a possible difference in the functional effects of FHM and familial epilepsy mutations of Nav1.1.

Recessive Loss-of-Function Mutation in the Pacemaker HCN2 Channel Causing Increased Neuronal Excitability in a Patient with Idiopathic Generalized Epilepsy

The hyperpolarization-activated Ih current, coded for by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, controls synaptic integration and intrinsic excitability in many brain areas. Because of their role in pacemaker function, defective HCN channels are natural candidates for contributing to epileptogenesis. Indeed, Ih is pathologically altered after experimentally induced seizures, and several independent data indicate a link between dysfunctional HCN channels and different forms of epilepsy. However, direct evidence for functional changes of defective HCN channels correlating with the disease in human patients is still elusive. By screening families with epilepsy for mutations in Hcn1 and Hcn2 genes, we found a recessive loss-of-function point mutation in the gene coding for the HCN2 channel in a patient with sporadic idiopathic generalized epilepsy. Of 17 screened members of the same family, the proband was the only one affected and homozygous for the mutation. The mutation (E515K) is located in the C-linker, a region known to affect channel gating. Functional analysis revealed that homomeric mutant, but not heteromeric wild-type/mutant channels, have a strongly inhibited function caused by a large negative shift of activation range and slowed activation kinetics, effectively abolishing the HCN2 contribution to activity. After transfection into acutely isolated newborn rat cortical neurons, homomeric mutant, but not heteromeric wild type/mutant channels, lowered the threshold of action potential firing and strongly increased cell excitability and firing frequency when compared with wild-type channels. This is the first evidence in humans for a single-point, homozygous loss-of-function mutation in HCN2 potentially associated with generalized epilepsy with recessive inheritance.

A caveolin-binding domain in the HCN4 channels mediates functional interaction with caveolin proteins

Pacemaker (HCN) channels have a key role in the generation and modulation of spontaneous activity of sinoatrial node myocytes. Previous work has shown that compartmentation of HCN4 pacemaker channels within caveolae regulates important functions, but the molecular mechanism responsible is still unknown. HCN channels have a conserved caveolin-binding domain (CBD) composed of three aromatic amino acids at the N-terminus; we sought to evaluate the role of this CBD in channel-protein interaction by mutational analysis. We generated two HCN4 mutants with a disrupted CBD (Y259S, F262V) and two with conservative mutations (Y259F, F262Y). In CHO cells expressing endogenous caveolin-1 (cav-1), alteration of the CBD shifted channels activation to more positive potentials, slowed deactivation and made Y259S and F262V mutants insensitive to cholesterol depletion-induced caveolar disorganization. CBD alteration also caused a significant decrease of current density, due to a weaker HCN4-cav-1 interaction and accumulation of cytoplasmic channels. These effects were absent in mutants with a preserved CBD. In caveolin-1-free fibroblasts, HCN4 trafficking was impaired and current density reduced with all constructs; the activation curve of F262V was not altered relative to wt, and that of Y259S displayed only half the shift than in CHO cells. The conserved CBD present in all HCN isoforms mediates their functional interaction with caveolins. The elucidation of the molecular details of HCN4-cav-1 interaction can provide novel information to understand the basis of cardiac phenotypes associated with some forms of caveolinopathies.

The X-linked intellectual disability protein IL1RAPL1 regulates dendrite complexity

Mutations and deletions of the interleukin-1 receptor accessory protein like 1 (IL1RAPL1) gene, located on the X chromosome, are associated with intellectual disability (ID) and autism spectrum disorder (ASD). IL1RAPL1 protein is located at the postsynaptic compartment of excitatory synapses and plays a role in synapse formation and stabilization. Here, using primary neuronal cultures and Il1rapl1-KO mice, we characterized the role of IL1RAPL1 in regulating dendrite morphology. In Il1rapl1-KO mice we identified an increased number of dendrite branching points in CA1 and CA2 hippocampal neurons associated to hippocampal cognitive impairment. Similarly, induced pluripotent stem cell-derived neurons from a patient carrying a null mutation of the IL1RAPL1 gene had more dendrites. In hippocampal neurons, the overexpression of full-length IL1RAPL1 and mutants lacking part of C-terminal domains leads to simplified neuronal arborization. This effect is abolished when we overexpressed mutants lacking part of N-terminal domains, indicating that the IL1RAPL1 extracellular domain is required for regulating dendrite development. We also demonstrate that PTPδ interaction is not required for this activity, while IL1RAPL1 mediates the activity of IL-1β on dendrite morphology. Our data reveal a novel specific function for IL1RAPL1 in regulating dendrite morphology that can help clarify how changes in IL1RAPL1-regulated pathways can lead to cognitive disorders in humans. SIGNIFICANCE STATEMENT Abnormalities in the architecture of dendrites have been observed in a variety of neurodevelopmental, neurodegenerative, and neuropsychiatric disorders. Here we show that the X-linked intellectual disability protein interleukin-1 receptor accessory protein like 1 (IL1RAPL1) regulates dendrite morphology of mice hippocampal neurons and induced pluripotent stem cell-derived neurons from a patient carrying a null mutation of IL1RAPL1 gene. We also found that the extracellular domain of IL1RAPL1 is required for this effect, independently of the interaction with PTPδ, but IL1RAPL1 mediates the activity of IL-1β on dendrite morphology. Our data reveal a novel specific function for IL1RAPL1 in regulating dendrite morphology that can help clarify how changes in IL1RAPL1-regulated pathways can lead to cognitive disorders in humans.

The impact of genetic and experimental studies on classification and therapy of the epilepsies

Different types of epilepsy are associated with gene mutations, in which seizures can be the only symptom (genetic epilepsies) or be one of the elements of complex clinical pictures that are often progressive over time (epileptic or epileptogenic encephalopathies). In epileptogenic encephalopathies, epileptic seizures and other neurological and cognitive signs are symptoms of genetically determined neuropathological or neurochemical disorders. In epileptic encephalopathies, epileptic activity itself is thought to contribute to severe cognitive and behavioral impairments above and beyond what might be expected from the underlying pathology alone. The distinction is conceptually clear and clinically relevant, as the different categories have a different prognosis in terms of both epilepsy and associated neurological and cognitive picture, but the boundaries are sometimes difficult to define in the clinical practice. Here we review the genetic epilepsies from the clinician perspective. A monogenic inheritance has been defined only in a minority of idiopathic epilepsies making improper to rename genetic the category of idiopathic epilepsies, until the presumptive multigenic mechanism will be demonstrated. A search for gene mutations must be done in any patient with candidate genetic types of epilepsy or epileptic/epileptogenic encephalopathy (e.g. familial forms) to complete the diagnostic process, define the prognosis and optimize the therapy. Advanced methods are available to express the gene variant in experimental model systems and test its effect on the properties of the affected protein, on neuronal excitability and on phenotypes in model organisms, and may help in identifying treatments with compatible action mechanisms. The influence of genetic studies on epilepsy taxonomy is now a matter of discussion: their impact on the international classification of the epilepsies will hopefully be defined soon.