Clinical whole-genome sequencing in severe early-onset epilepsy reveals new genes and improves molecular diagnosis.

In severe early-onset epilepsy, precise clinical and molecular genetic diagnosis is complex, as many metabolic and electro-physiological processes have been implicated in disease causation. The clinical phenotypes share many features such as complex seizure types and developmental delay. Molecular diagnosis has historically been confined to sequential testing of candidate genes known to be associated with specific sub-phenotypes, but the diagnostic yield of this approach can be low. We conducted whole-genome sequencing (WGS) on six patients with severe early-onset epilepsy who had previously been refractory to molecular diagnosis, and their parents. Four of these patients had a clinical diagnosis of Ohtahara Syndrome (OS) and two patients had severe non-syndromic early-onset epilepsy (NSEOE). In two OS cases, we found de novo non-synonymous mutations in the genes KCNQ2 and SCN2A. In a third OS case, WGS revealed paternal isodisomy for chromosome 9, leading to identification of the causal homozygous missense variant in KCNT1, which produced a substantial increase in potassium channel current. The fourth OS patient had a recessive mutation in PIGQ that led to exon skipping and defective glycophosphatidyl inositol biosynthesis. The two patients with NSEOE had likely pathogenic de novo mutations in CBL and CSNK1G1, respectively. Mutations in these genes were not found among 500 additional individuals with epilepsy. This work reveals two novel genes for OS, KCNT1 and PIGQ. It also uncovers unexpected genetic mechanisms and emphasizes the power of WGS as a clinical tool for making molecular diagnoses, particularly for highly heterogeneous disorders.

Regulation of neuronal excitability by interaction of fragile X mental retardation protein with slack potassium channels.

Loss of the RNA-binding protein fragile X mental retardation protein (FMRP) represents the most common form of inherited intellectual disability. Studies with heterologous expression systems indicate that FMRP interacts directly with Slack Na(+)-activated K(+) channels (K(Na)), producing an enhancement of channel activity. We have now used Aplysia bag cell (BC) neurons, which regulate reproductive behaviors, to examine the effects of Slack and FMRP on excitability. FMRP and Slack immunoreactivity were colocalized at the periphery of isolated BC neurons, and the two proteins could be reciprocally coimmunoprecipitated. Intracellular injection of FMRP lacking its mRNA binding domain rapidly induced a biphasic outward current, with an early transient tetrodotoxin-sensitive component followed by a slowly activating sustained component. The properties of this current matched that of the native Slack potassium current, which was identified using an siRNA approach. Addition of FMRP to inside-out patches containing native Aplysia Slack channels increased channel opening and, in current-clamp recordings, produced narrowing of action potentials. Suppression of Slack expression did not alter the ability of BC neurons to undergo a characteristic prolonged discharge in response to synaptic stimulation, but prevented recovery from a prolonged inhibitory period that normally follows the discharge. Recovery from the inhibited period was also inhibited by the protein synthesis inhibitor anisomycin. Our studies indicate that, in BC neurons, Slack channels are required for prolonged changes in neuronal excitability that require new protein synthesis, and raise the possibility that channel-FMRP interactions may link changes in neuronal firing to changes in protein translation.

The N-terminal half of the connexin protein contains the core elements of the pore and voltage gates.

Connexins form channels with large aqueous pores that mediate fluxes of inorganic ions and biological signaling molecules. Studies aimed at identifying the connexin pore now include a crystal structure that provides details of putative pore-lining residues that need to be verified using independent biophysical approaches. Here we extended our initial cysteine-scanning studies of the TM1/E1 region of Cx46 hemichannels to include TM2 and TM3 transmembrane segments. No evidence of reactivity was observed in either TM2 or TM3 probed with small or large thiol-modifying reagents. Several identified pore residues in E1 of Cx46 have been verified in different Cx isoforms. Use of variety of thiol reagents indicates that the connexin hemichannel pore is large and flexible enough, at least in the extracellular part of the pore funnel, to accommodate uncommonly large side chains. We also find that that gating characteristics are largely determined by the same domains that constitute the pore. These data indicate that biophysical and structural studies are converging towards a view that the N-terminal half of the Cx protein contains the principal components of the pore and gating elements, with NT, TM1 and E1 forming the pore funnel.

Fragile X mental retardation protein is required for rapid experience-dependent regulation of the potassium channel Kv3.1b.

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates synaptic plasticity by repressing translation of specific mRNAs. We found that FMRP binds mRNA encoding the voltage-gated potassium channel Kv3.1b in brainstem synaptosomes. To explore the regulation of Kv3.1b by FMRP, we investigated Kv3.1b immunoreactivity and potassium currents in the auditory brainstem sound localization circuit of male mice. The unique features of this circuit allowed us to control neuronal activity in vivo by exposing animals to high-frequency, amplitude-modulated stimuli, which elicit predictable and stereotyped patterns of input to the anterior ventral cochlear nucleus (AVCN) and medial nucleus of the trapezoid body (MNTB). In wild-type (WT) animals, Kv3.1b is expressed along a tonotopic gradient in the MNTB, with highest levels in neurons at the medial, high-frequency end. At baseline, Fmr1(-/-) mice, which lack FMRP, displayed dramatically flattened tonotopicity in Kv3.1b immunoreactivity and K(+) currents relative to WT controls. Moreover, after 30 min of acoustic stimulation, levels of Kv3.1b immunoreactivity were significantly elevated in both the MNTB and AVCN of WT, but not Fmr1(-/-), mice. These results suggest that FMRP is necessary for maintenance of the gradient in Kv3.1b protein levels across the tonotopic axis of the MNTB, and are consistent with a role for FMRP as a repressor of protein translation. Using numerical simulations, we demonstrate that Kv3.1b tonotopicity may be required for accurate encoding of stimulus features such as modulation rate, and that disruption of this gradient, as occurs in Fmr1(-/-) animals, degrades processing of this information.

The N-terminal domain of Slack determines the formation and trafficking of Slick/Slack heteromeric sodium-activated potassium channels.

Potassium channels activated by intracellular Na(+) ions (K(Na)) play several distinct roles in regulating the firing patterns of neurons, and, at the single channel level, their properties are quite diverse. Two known genes, Slick and Slack, encode K(Na) channels. We have now found that Slick and Slack subunits coassemble to form heteromeric channels that differ from the homomers in their unitary conductance, kinetic behavior, subcellular localization, and response to activation of protein kinase C. Heteromer formation requires the N-terminal domain of Slack-B, one of the alternative splice variants of the Slack channel. This cytoplasmic N-terminal domain of Slack-B also facilitates the localization of heteromeric K(Na) channels to the plasma membrane. Immunocytochemical studies indicate that Slick and Slack-B subunits are coexpressed in many central neurons. Our findings provide a molecular explanation for some of the diversity in reported properties of neuronal K(Na) channels.

Amino-termini isoforms of the Slack K+ channel, regulated by alternative promoters, differentially modulate rhythmic firing and adaptation.

The rates of activation and unitary properties of Na+-activated K+ (K(Na)) currents have been found to vary substantially in different types of neurones. One class of K(Na) channels is encoded by the Slack gene. We have now determined that alternative RNA splicing gives rise to at least five different transcripts for Slack, which produce Slack channels that differ in their predicted cytoplasmic amino-termini and in their kinetic properties. Two of these, termed Slack-A channels, contain an amino-terminus domain closely resembling that of another class of K(Na) channels encoded by the Slick gene. Neuronal expression of Slack-A channels and of the previously described Slack isoform, now called Slack-B, are driven by independent promoters. Slack-A mRNAs were enriched in the brainstem and olfactory bulb and detected at significant levels in four different brain regions. When expressed in CHO cells, Slack-A channels activate rapidly upon depolarization and, in single channel recordings in Xenopus oocytes, are characterized by multiple subconductance states with only brief transient openings to the fully open state. In contrast, Slack-B channels activate slowly over hundreds of milliseconds, with openings to the fully open state that are approximately 6-fold longer than those for Slack-A channels. In numerical simulations, neurones in which outward currents are dominated by a Slack-A-like conductance adapt very rapidly to repeated or maintained stimulation over a wide range of stimulus strengths. In contrast, Slack-B currents promote rhythmic firing during maintained stimulation, and allow adaptation rate to vary with stimulus strength. Using an antibody that recognizes all amino-termini isoforms of Slack, Slack immunoreactivity is present at locations that have no Slack-B-specific staining, including olfactory bulb glomeruli and the dendrites of hippocampal neurones, suggesting that Slack channels with alternate amino-termini such as Slack-A channels are present at these locations. Our data suggest that alternative promoters of the Slack gene differentially modulate the properties of neurones.

Regulation of connexin hemichannels by monovalent cations.

Opening of connexin hemichannels in the plasma membrane is highly regulated. Generally, depolarization and reduced extracellular Ca2+ promote hemichannel opening. Here we show that hemichannels formed of Cx50, a principal lens connexin, exhibit a novel form of regulation characterized by extraordinary sensitivity to extracellular monovalent cations. Replacement of extracellular Na+ with K+, while maintaining extracellular Ca2+ constant, resulted in >10-fold potentiation of Cx50 hemichannel currents, which reversed upon returning to Na+. External Cs+, Rb+, NH4+, but not Li+, choline, or TEA, exhibited a similar effect. The magnitude of potentiation of Cx50 hemichannel currents depended on the concentration of extracellular Ca2+, progressively decreasing as external Ca2+ was reduced. The primary effect of K+ appears to be a reduction in the ability of Ca2+, as well as other divalent cations, to close Cx50 hemichannels. Cx46 hemichannels exhibited a modest increase upon substituting Na+ with K+. Analyses of reciprocal chimeric hemichannels that swap NH2- and COOH-terminal halves of Cx46 and Cx50 demonstrate that the difference in regulation by monovalent ions in these connexins resides in the NH2-terminal half. Connexin hemichannels have been implicated in physiological roles, e.g., release of ATP and NAD+ and in pathological roles, e.g., cell death through loss or entry of ions and signaling molecules. Our results demonstrate a new, robust means of regulating hemichannels through a combination of extracellular monovalent and divalent cations, principally Na+, K+, and Ca2+.

Stimulation of Slack K(+) Channels Alters Mass at the Plasma Membrane by Triggering Dissociation of a Phosphatase-Regulatory Complex.

Human mutations in the cytoplasmic C-terminal domain of Slack sodium-activated potassium (KNa) channels result in childhood epilepsy with severe intellectual disability. Slack currents can be increased by pharmacological activators or by phosphorylation of a Slack C-terminal residue by protein kinase C. Using an optical biosensor assay, we find that Slack channel stimulation in neurons or transfected cells produces loss of mass near the plasma membrane. Slack mutants associated with intellectual disability fail to trigger any change in mass. The loss of mass results from the dissociation of the protein phosphatase 1 (PP1) targeting protein, Phactr-1, from the channel. Phactr1 dissociation is specific to wild-type Slack channels and is not observed when related potassium channels are stimulated. Our findings suggest that Slack channels are coupled to cytoplasmic signaling pathways and that dysregulation of this coupling may trigger the aberrant intellectual development associated with specific childhood epilepsies.

Human slack potassium channel mutations increase positive cooperativity between individual channels.

Disease-causing mutations in ion channels generally alter intrinsic gating properties such as activation, inactivation, and voltage dependence. We examined nine different mutations of the KCNT1 (Slack) Na(+)-activated K(+) channel that give rise to three distinct forms of epilepsy. All produced many-fold increases in current amplitude compared to the wild-type channel. This could not be accounted for by increases in the intrinsic open probability of individual channels. Rather, greatly increased opening was a consequence of cooperative interactions between multiple channels in a patch. The degree of cooperative gating was much greater for all of the mutant channels than for the wild-type channel, and could explain increases in current even in a mutant with reduced unitary conductance. We also found that the same mutation gave rise to different forms of epilepsy in different individuals. Our findings indicate that a major consequence of these mutations is to alter channel-channel interactions.

De novo gain-of-function KCNT1 channel mutations cause malignant migrating partial seizures of infancy.

Malignant migrating partial seizures of infancy (MMPSI) is a rare epileptic encephalopathy of infancy that combines pharmacoresistant seizures with developmental delay. We performed exome sequencing in three probands with MMPSI and identified de novo gain-of-function mutations affecting the C-terminal domain of the KCNT1 potassium channel. We sequenced KCNT1 in 9 additional individuals with MMPSI and identified mutations in 4 of them, in total identifying mutations in 6 out of 12 unrelated affected individuals. Functional studies showed that the mutations led to constitutive activation of the channel, mimicking the effects of phosphorylation of the C-terminal domain by protein kinase C. In addition to regulating ion flux, KCNT1 has a non-conducting function, as its C terminus interacts with cytoplasmic proteins involved in developmental signaling pathways. These results provide a focus for future diagnostic approaches and research for this devastating condition.

A less flexible BK channel opens more easily.

BK potassium channels regulate spike width and firing rate. A BK point mutation in humans enhances channel activity, leading to epilepsy. In this issue of Neuron, Yang and colleagues use this mutation to demonstrate that channel opening is determined by the flexibility of a region that contains a Ca(2+)-binding site.

Fragile X mental retardation protein controls gating of the sodium-activated potassium channel Slack.

In humans, the absence of Fragile X mental retardation protein (FMRP), an RNA-binding protein, results in Fragile X syndrome, the most common inherited form of intellectual disability. Using biochemical and electrophysiological studies, we found that FMRP binds to the C terminus of the Slack sodium-activated potassium channel to activate the channel in mice. Our findings suggest that Slack activity provides a link between patterns of neuronal firing and changes in protein translation.

Protein kinase C modulates inactivation of Kv3.3 channels.

Modulation of some Kv3 family potassium channels by protein kinase C (PKC) regulates their amplitude and kinetics and adjusts firing patterns of auditory neurons in response to stimulation. Nevertheless, little is known about the modulation of Kv3.3, a channel that is widely expressed throughout the nervous system and is the dominant Kv3 family member in auditory brainstem. We have cloned the cDNA for the Kv3.3 channel from mouse brain and have expressed it in a mammalian cell line and in Xenopus oocytes to characterize its biophysical properties and modulation by PKC. Kv3.3 currents activate at positive voltages and undergo inactivation with time constants of 150-250 ms. Activators of PKC increased current amplitude and removed inactivation of Kv3.3 currents, and a specific PKC pseudosubstrate inhibitor peptide prevented the effects of the activators. Elimination of the first 78 amino acids of the N terminus of Kv3.3 produced noninactivating currents suggesting that PKC modulates N-type inactivation, potentially by phosphorylation of sites in this region. To identify potential phosphorylation sites, we investigated the response of channels in which serines in this N-terminal domain were subjected to mutagenesis. Our results suggest that serines at positions 3 and 9 are potential PKC phosphorylation sites. Computer simulations of model neurons suggest that phosphorylation of Kv3.3 by PKC may allow neurons to maintain action potential height during stimulation at high frequencies, and may therefore contribute to stimulus-induced changes in the intrinsic excitability of neurons such as those of the auditory brainstem.

Diabetes-induced changes in the alternative splicing of the slo gene in corporal tissue.

OBJECTIVES: Erectile dysfunction is a common diabetic complication. Preclinical studies have documented that the Slo gene (encoding the BK or Maxi-K channel alpha-subunit) plays a critical role in erectile function. Therefore, we determined whether diabetes induces changes in the splicing of the Slo gene relevant to erectile function. METHODS: Reverse transcriptase-polymerase chain reaction was used to compare Slo splice variant expression in corporal tissue excised from control and streptozotocin (STZ)-induced diabetic Fischer F-344 rats. Splice variants were sequenced, characterized by patch clamping, and fused to green fluorescent protein to determine cellular localization. The impact of altered Slo expression on erectile function was further evaluated in vivo. RESULTS: A novel Slo splice variant (SVcyt, with a cytoplasmic location) was predominantly expressed in corporal tissue from control rats. STZ-diabetes caused upregulation of a channel-forming transcript SV0. Preliminary results suggest that SV0 was also more prevalent in the corporal tissue of human diabetic compared with nondiabetic patients. The change in isoform expression in STZ-treated rats was partially reversed by insulin treatment. Intracorporal injection of a plasmid expressing the SV0 transcript, but not SVcyt, restored erectile function in STZ-diabetic rats. CONCLUSIONS: Alternative splicing of the Slo transcript may represent an important compensatory mechanism to increase the ease with which relaxation of corporal tissue may be triggered as a result of a diabetes-related decline in erectile capacity.

Correlative studies of gating in Cx46 and Cx50 hemichannels and gap junction channels.

Transjunctional voltage (V(j)) gating of gap junction (GJ) channels formed of connexins has been proposed to occur by gating of the component hemichannels. We took advantage of the ability of Cx46 and Cx50 to function as unapposed hemichannels to identify gating properties intrinsic to hemichannels and how they contribute to gating of GJ channels. We show that Cx46 and Cx50 hemichannels contain two distinct gating mechanisms that generate reductions in conductance for both membrane polarities. At positive voltages, gating is similar in Cx46 and Cx50 hemichannels, primarily showing increased transitioning to long-lived substates. At negative voltages, Cx46 currents deactivate completely and the underlying single hemichannels exhibit transitions to a fully closed state. In contrast, Cx50 currents do not deactivate completely at negative voltages and the underlying single hemichannels predominantly exhibit transitions to various substates. Transitions to a fully closed state occur, but are infrequent. In the respective GJ channels, both forms of gating contribute to the reduction in conductance by V(j). However, examination of gating of mutant hemichannels and GJ channels in which the Asp at position 3 was replaced with Asn (D3N) showed that the positive hemichannel gate predominantly closes Cx50 GJs, whereas the negative hemichannel gate predominantly closes Cx46 GJs in response to V(j). We also report, for the first time, single Cx50 hemichannels in oocytes to be inwardly rectifying, high conductance channels (gamma = 470 pS). The antimalarial drug mefloquine, which selectively blocks Cx50 and not Cx46 GJs, shows the same selectivity in Cx50 and Cx46 hemichannels indicating that the actions of such uncoupling agents, like voltage gating, are intrinsic hemichannel properties.