Mitchell Goldfarb

Voltage-gated sodium channels undergo reversible voltage/time-dependent transitions from closed to open and inactivated states. The voltage setpoints and efficiency of cardiac sodium channel Nav1.5 state transitions are crucial for tuning the initiation and conduction of myocardial action potentials. The channel’s cytoplasmic carboxyl-terminal domain (CTD) regulates gating by intramolecular interactions and by serving as a hub for the binding of accessory proteins. We have investigated the roles of the CTD in intrinsic and FGF homologous factor (FHF)-modulated Nav1.5 gating through structure-guided CTD subdomain mutagenesis. The EF-hand module within the CTD was found to exert the most profound effects on channel gating, strongly influencing voltage dependence of inactivation and activation, accelerating inactivation from the closed state, decelerating inactivation from the open state, minimizing persistent sodium current, and serving as the binding domain for FHF proteins. Nav1.5D1788K bearing a missense mutation in the EF-hand motif displayed a depolarizing shift in voltage dependence of activation and generated enhanced persistent sodium current without altering the voltage dependence of channel inactivation. Another EF-hand mutant, Nav1.5L1861A, underwent closed-state inactivation at more negative membrane potential and at an accelerated rate but did not display other phenotypes associated with CTD deletion. Missense mutation Nav1.5V1776A in the juxtamembrane region between the EF-hand and the channel pore helices did not alter intrinsic gating properties but impaired FHF modulation of inactivation gating. Our channel physiology studies, together with the prior structural data from others, suggest that the voltage and rate of channel inactivation from the closed state are governed by an intramolecular hydrophobic interaction of the CTD EF-hand with the cytoplasmic inactivation loop helix and the extension of this binding interface upon FHF-induced restructuring of the juxtamembrane region. The CTD also tunes voltage-dependent activation and helps minimize persistent sodium current through distinct, presumed electrostatic mechanisms.

Voltage-gated sodium channels undergo reversible voltage/time-dependent transitions from closed to open and inactivated states. The voltage setpoints and efficiency of cardiac sodium channel Na _v 1.5 state transitions are crucial for tuning the initiation and conduction of myocardial action potentials. The channel’s cytoplasmic carboxyl-terminal domain (CTD) regulates gating by intramolecular interactions and by serving as a hub for the binding of accessory proteins. We have investigated the roles of the CTD in intrinsic and FGF homologous factor (FHF)-modulated Na _v 1.5 gating through structure-guided CTD subdomain mutagenesis. The EF-hand module within the CTD was found to exert the most profound effects on channel gating, strongly influencing voltage-dependence of inactivation and activation, accelerating inactivation from the closed state, decelerating inactivation from the open state, minimizing persistent sodium current, and serving as the binding domain for FHF proteins. Na _v 1.5 ^D1788K bearing a missense mutation in the EF-hand motif displayed a depolarizing shift in voltage dependence of activation and generated greatly enhanced persistent sodium current without altering the voltage dependence of channel inactivation. Reciprocally, Na _v 1.5 ^L1861A bearing a different missense mutation in the EF-hand underwent closed-state inactivation at more negative membrane potential and at an accelerated rate, but did not display other phenotypes associated with CTD deletion. Na _v 1.5 ^{V1776A/T1778A} bearing mutations in the juxtamembrane region between the EF-hand and the channel pore helices displayed wild-type intrinsic gating properties, while FHF modulation of inactivation gating was impaired. Our channel physiology studies together with prior structural data suggest that the voltage and rate of channel inactivation from the closed state are governed by an intramolecular hydrophobic interaction of the CTD EF-hand with the cytoplasmic inactivation loop helix and the extension of this binding interface upon FHF-induced restructuring of the juxtamembrane region, while a distinct CTD intramolecular electrostatic interaction modulates voltage-dependent activation and minimizes persistent sodium current.

Since their discovery nearly 30 years ago, fibroblast growth factor homologous factors (FHFs) are now known to control the functionality of excitable tissues through a range of mechanisms. Nervous and cardiac system dysfunctions are caused by loss‐ or gain‐of‐function mutations in FHF genes. The best understood ‘canonical’ targets for FHF action are voltage‐gated sodium channels, and recent studies have expanded the repertoire of ways that FHFs modulate sodium channel gating. Additional ‘non‐canonical’ functions of FHFs in excitable and non‐excitable cells, including cancer cells, have been reported over the past dozen years. This review summarizes and evaluates reported canonical and non‐canonical FHF functions. image

Transient voltage-gated sodium currents are essential for the initiation and conduction of action potentials in neurons and cardiomyocytes. The amplitude and duration of sodium currents are tuned by intracellular fibroblast growth factor homologous factors (FHFs/iFGFs) that associate with the cytoplasmic tails of voltage-gated sodium channels (Na_vs), and genetic ablation of Fhf genes disturbs neurological and cardiac functions. Among reported phenotypes, Fhf2 ^null mice undergo lethal hyperthermia-induced cardiac conduction block attributable to the combined effects of FHF2 deficiency and elevated temperature on the cardiac sodium channel (Na_v1.5) inactivation rate. Fhf2 ^null mice also display a lack of heat nociception, while retaining other somatosensory capabilities. Here, we use electrophysiological and computational methods to show that the heat nociception deficit can be explained by the combined effects of elevated temperature and FHF2 deficiency on the fast inactivation gating of Na_v1.7 and tetrodotoxin-resistant sodium channels expressed in dorsal root ganglion C fibers. Hence, neurological and cardiac heat-associated deficits in Fhf2 ^null mice derive from shared impacts of FHF deficiency and temperature towards Na_v inactivation gating kinetics in distinct tissues.

Transient voltage-gated sodium currents are essential for the initiation and conduction of action potentials in neurons and cardiomyocytes. The amplitude and duration of sodium currents are tuned by intracellular fibroblast growth factor homologous factors (FHFs/iFGFs) that associate with the cytoplasmic tails of voltage-gated sodium channels (Na _v s), and genetic ablation of Fhf genes disturbs neurological and cardiac functions. Among reported phenotypes, Fhf2 ^null mice undergo lethal hyperthermia-induced cardiac conduction block attributable to the combined effects of FHF2 deficiency and elevated temperature on the cardiac sodium channel (Na _v 1.5) inactivation rate. Fhf2 ^null mice also display a lack of heat nociception, while retaining other somatosensory capabilities. Here, we use electrophysiological and computational methods to show that the heat nociception deficit can be explained by the combined effects of elevated temperature and FHF2 deficiency on the fast inactivation gating of Na _v 1.7 and tetrodotoxin-resistant sodium channels expressed in dorsal root ganglion C fibers. Hence, neurological and cardiac heat-associated deficits in Fhf2 ^null mice derive from shared impacts of FHF deficiency and temperature towards Na _v inactivation gating kinetics in distinct tissues.

Summary: Objective: Fibroblast growth factor homologous factors (FHFs) are brain and cardiac sodium channel‐binding proteins that modulate channel density and inactivation gating. A recurrent de novo gain‐of‐function missense mutation in the FHF1(FGF12) gene (p.Arg52His) is associated with early infantile epileptic encephalopathy 47 (EIEE47; Online Mendelian Inheritance in Man database 617166). To determine whether the FHF1 missense mutation is sufficient to cause EIEE and to establish an animal model for EIEE47, we sought to engineer this mutation into mice.Methods: The Arg52His mutation was introduced into fertilized eggs by CRISPR (clustered regularly interspaced short palindromic repeats) editing to generate Fhf1^R52H/F+ mice. Spontaneous epileptiform events in Fhf1^R52H/+ mice were assessed by cortical electroencephalography (EEG) and video monitoring. Basal heart rhythm and seizure‐induced arrhythmia were recorded by electrocardiography. Modulation of cardiac sodium channel inactivation by FHF1B_R52H protein was assayed by voltage‐clamp recordings of FHF‐deficient mouse cardiomyocytes infected with adenoviruses expressing wild‐type FHF1B or FHF1B_R52H protein.Results: All Fhf1^R52H/+ mice experienced seizure or seizurelike episodes with lethal ending between 12 and 26 days of age. EEG recordings in 19–20‐day‐old mice confirmed sudden unexpected death in epilepsy (SUDEP) as severe tonic seizures immediately preceding loss of brain activity and death. Within 2–53 s after lethal seizure onset, heart rate abruptly declined from 572 ± 16 bpm to 108 ± 15 bpm, suggesting a parasympathetic surge accompanying seizures that may have contributed to SUDEP. Although ectopic overexpression of FHF1B_R52H in cardiomyocytes induced a 15‐mV depolarizing shift in voltage of steady‐state sodium channel inactivation and slowed the rate of channel inactivation, heart rhythm was normal in Fhf1^R52H/+ mice prior to seizure.Significance: The Fhf1 missense mutation p.Arg52His induces epileptic encephalopathy with full penetrance in mice. Both Fhf1 (p.Arg52His) and Scn8a (p.Asn1768Asp) missense mutations enhance sodium channel Na_v1.6 currents and induce SUDEP with bradycardia in mice, suggesting an FHF1/Na_v1.6 functional axis underlying altered brain sodium channel gating in epileptic encephalopathy.

Rationale: FHFs (fibroblast growth factor homologous factors) are key regulators of sodium channel (Na _V ) inactivation. Mutations in these critical proteins have been implicated in human diseases including Brugada syndrome, idiopathic ventricular arrhythmias, and epileptic encephalopathy. The underlying ionic mechanisms by which reduced Na _v availability in Fhf2 knockout ( Fhf2 ^KO ) mice predisposes to abnormal excitability at the tissue level are not well defined. Objective: Using animal models and theoretical multicellular linear strands, we examined how FHF2 orchestrates the interdependency of sodium, calcium, and gap junctional conductances to safeguard cardiac conduction. Methods and Results: Fhf2 ^KO mice were challenged by reducing calcium conductance (gCa _V ) using verapamil or by reducing gap junctional conductance (Gj) using carbenoxolone or by backcrossing into a cardiomyocyte-specific Cx43 (connexin 43) heterozygous background. All conditions produced conduction block in Fhf2 ^KO mice, with Fhf2 wild-type ( Fhf2 ^WT ) mice showing normal impulse propagation. To explore the ionic mechanisms of block in Fhf2 ^KO hearts, multicellular linear strand models incorporating FHF2-deficient Na _v inactivation properties were constructed and faithfully recapitulated conduction abnormalities seen in mutant hearts. The mechanisms of conduction block in mutant strands with reduced gCa _V or diminished Gj are very different. Enhanced Na _v inactivation due to FHF2 deficiency shifts dependence onto calcium current (I _Ca ) to sustain electrotonic driving force, axial current flow, and action potential (AP) generation from cell-to-cell. In the setting of diminished Gj, slower charging time from upstream cells conspires with accelerated Na _v inactivation in mutant strands to prevent sufficient downstream cell charging for AP propagation. Conclusions: FHF2-dependent effects on Na _v inactivation ensure adequate sodium current (I _Na ) reserve to safeguard against numerous threats to reliable cardiac impulse propagation.

Autism spectrum disorders (ASD) are pervasive neurodevelopmental conditions that often involve mutations affecting synaptic mechanisms. Recently, the involvement of cerebellum in ASD has been suggested but the underlying functional alterations remained obscure. We investigated single-neuron and microcircuit properties in IB2 KO mice, which present a cerebellar phenotype associated with ASD. Granule cells showed a larger NMDA receptor-mediated current and enhanced intrinsic excitability raising the excitatory/inhibitory balance. Furthermore, the spatial organization of granular layer responses to mossy fibers shifted from a Mexican hat to stovepipe hat profile, with stronger excitation in the core and weaker inhibition in the surround. Finally, the size and extension of long-term synaptic plasticity was remarkably increased. These results show for the first time that hyper-excitability and hyper-plasticity disrupt signal transfer in the granular layer of IB2 KO mice supporting cerebellar involvement in the pathogenesis of ASD.

FGF signaling, an important component of intercellular communication, is required in many tissues throughout development to promote diverse cellular processes. Whether FGF receptors (FGFRs) accomplish such varied tasks in part by activating different intracellular transducers in different contexts remains unclear. Here, we used the developing mouse telencephalon as an example to study the role of the FRS adapters FRS2 and FRS3 in mediating the functions of FGFRs. Using tissue-specific and germline mutants, we examined the requirement of Frs genes in two FGFR-dependent processes. We found that Frs2 and Frs3 are together required for the differentiation of a subset of medial ganglionic eminence (MGE)-derived neurons, but are dispensable for the survival of early telencephalic precursor cells, in which any one of three FGFRs (FGFR1, FGFR2, or FGFR3) is sufficient for survival. Although FRS adapters are dispensable for ERK-1/2 activation, they are required for AKT activation within the subventricular zone of the developing MGE. Using an FRS2,3-binding site mutant of Fgfr1, we established that FRS adapters are necessary for mediating most or all FGFR1 signaling, not only in MGE differentiation, but also in cell survival, implying that other adapters mediate at least in part the signaling from FGFR2 and FGFR3. Our study provides an example of a contextual role for an intracellular transducer and contributes to our understanding of how FGF signaling plays diverse developmental roles. SIGNIFICANCE STATEMENT FGFs promote a range of developmental processes in many developing tissues and at multiple developmental stages. The mechanisms underlying this multifunctionality remain poorly defined in vivo. Using telencephalon development as an example, we show here that FRS adapters exhibit some selectivity in their requirement for mediating FGF receptor (FGFR) signaling and activating downstream mediators that depend on the developmental process, with a requirement in neuronal differentiation but not cell survival. Differential engagement of FRS and non-FRS intracellular adapters downstream of FGFRs could therefore in principle explain how FGFs play several distinct roles in other developing tissues and developmental stages.