Carlos G. Vanoye

Molecular basis of an inherited epilepsy

Epilepsy is a common neurological condition that reflects neuronal hyperexcitability arising from largely unknown cellular and molecular mechanisms. In generalized epilepsy with febrile seizures plus, an autosomal dominant epilepsy syndrome, mutations in three genes coding for voltage-gated sodium channel alpha or beta1 subunits (SCN1A, SCN2A, SCN1B) and one GABA receptor subunit gene (GABRG2) have been identified. Here, we characterize the functional effects of three mutations in the human neuronal sodium channel alpha subunit SCN1A by heterologous expression with its known accessory subunits, beta1 and beta2, in cultured mammalian cells. SCN1A mutations alter channel inactivation, resulting in persistent inward sodium current. This gain-of-function abnormality will likely enhance excitability of neuronal membranes by causing prolonged membrane depolarization, a plausible underlying biophysical mechanism responsible for this inherited human epilepsy.

Nucleotides and phospholipids compete for binding to the C terminus of KATP channels.

Inwardly rectifying, ATP-sensitive K+ channels (KATP) couple metabolism to either cell excitability (Kir6.x) or potassium secretion (Kir1.1). Phosphatidylinositol phospholipids, like PI(4,5)P2, antagonize nucleotide inhibition of KATP channels enhancing the coupling of metabolic events to cell electrical or transport activity. The mechanism by which phospholipids relieve ATP block is unclear. We have shown that maltose-binding fusion proteins (MBP) containing the COOH termini of KATP channels (Kir1.1, Kir6.1, and Kir6.2) form functional tetramers that directly bind at least two ATP molecules with negative cooperativity. Here we show that purified phosphatidylinositol phospholipids compete for 2,4,6,-trinitrophenyl (TNP)-ATP binding to the COOH termini of KATP channels with EC50 values for PIP2 between 6–8 μM. The phospholipid potency profile was PIP3 > PIP2 = PIP > PI, suggesting that net phospholipid charge was important. A role for head group charge was supported by polycations (neomycin, spermine, and polylysine) reversing the effect of PIP2 on TNP-ATP binding to the Kir1.1 channel COOH terminal fusion protein. In contrast, the water-soluble charged hydrolytic product of PIP2, inositol(1,4,5)P3 (IP3), had no effect on TNP-ATP binding, suggesting that the acyl chain of PIP2 was also necessary for its effect on TNP-ATP binding. Indeed, neutral and charged lipids had weak, but significant, effects on TNP-ATP binding. Whereas μM concentrations of PIP2 could compete with TNP-ATP, we found that mM concentrations of MgATP were required to compete with PIP2 for binding to these KATP channel COOH termini. Thus the COOH termini of KATP channels form a nucleotide- and phospholipid-modulated channel gate on which ATP and phospholipids compete for binding.

Sodium channel dysfunction in intractable childhood epilepsy with generalized tonic-clonic seizures.

Mutations in SCN1A, the gene encoding the brain voltage‐gated sodium channel α1 subunit (NaV1.1), are associated with genetic forms of epilepsy, including generalized epilepsy with febrile seizures plus (GEFS+ type 2), severe myoclonic epilepsy of infancy (SMEI) and related conditions. Several missense SCN1A mutations have been identified in probands affected by the syndrome of intractable childhood epilepsy with generalized tonic–clonic seizures (ICEGTC), which bears similarity to SMEI. To test whether ICEGTC arises from molecular mechanisms similar to those involved in SMEI, we characterized eight ICEGTC missense mutations by whole‐cell patch clamp recording of recombinant human SCN1A heterologously expressed in cultured mammalian cells. Two mutations (G979R and T1709I) were non‐functional. The remaining alleles (T808S, V983A, N1011I, V1611F, P1632S and F1808L) exhibited measurable sodium current, but had heterogeneous biophysical phenotypes. Mutant channels exhibited lower (V983A, N1011I and F1808L), greater (T808S) or similar (V1611F and P1632S) peak sodium current densities compared with wild‐type (WT) SCN1A. Three mutations (V1611F, P1632S and F1808L) displayed hyperpolarized conductance–voltage relationships, while V983A exhibited a strong depolarizing shift in the voltage dependence of activation. All mutants except T808S had hyperpolarized shifts in the voltage dependence of steady‐state channel availability. Three mutants (V1611F, P1632S and F1808L) exhibited persistent sodium current ranging from ∼1–3% of peak current amplitude that was significantly greater than WT‐SCN1A. Several mutants had impaired slow inactivation, with V983A showing the most prominent effect. Finally, all of the functional alleles exhibited reduced use‐dependent channel inhibition. In summary, SCN1A mutations associated with ICEGTC result in a wide spectrum of biophysical defects, including mild‐to‐moderate gating impairments, shifted voltage dependence and reduced use dependence. The constellation of biophysical abnormalities for some mutants is distinct from those previously observed for GEFS+ and SMEI, suggesting possible, but complex, genotype–phenotype correlations.

Functional characterization of recombinant human ClC-4 chloride channels in cultured mammalian cells

Members of the ClC chloride channel family participate in several physiological processes and are linked to human genetic diseases. The physiological role of ClC‐4 is unknown and previous detailed characterizations of recombinant human ClC‐4 (hClC‐4) have provided conflicting results. To re‐examine the hClC‐4 phenotype, recombinant hClC‐4 was expressed in three distinct mammalian cell lines and characterized using patch‐clamp techniques. In all cells, the expression of hClC‐4 generated strongly outward‐rectifying Cl− currents with the conductance sequence: SCN−≫ NO3−≫Cl− > Br−≈ I−≫ aspartate. Continuous activity of hClC‐4 was sustained to different degrees by internal nucleotides: ATP ≈ ATPγS ≫ AMP‐PNP ≈ GTP > ADP. Although non‐hydrolysable nucleotides are sufficient for channel function, ATP hydrolysis is required for full activity. Changing the extracellular (2 mm or nominal Ca2+‐free) or intracellular Ca2+ (25 or 250 nm) concentration did not alter hClC‐4 currents. Acidification of external pH (pHo) inhibited hClC‐4 currents (half‐maximal inhibition ≈ 6.19), whereas neither external alkalinization to pH 8.4 nor internal acidification to pH 6.0 reduced current levels. Single‐channel recordings demonstrated a Cl− channel active only at depolarizing potentials with a slope conductance of ∼3 pS. Acidic pHo did not alter single‐channel conductance. We conclude that recombinant hClC‐4 encodes a small‐conductance, nucleotide‐dependent, Ca2+‐independent outward‐rectifying chloride channel that is inhibited by external acidification. This detailed characterization will be highly valuable in comparisons of hClC‐4 function with native chloride channel activities and for future structure‐function correlations.

De novo KCNB1 mutations in epileptic encephalopathy

Numerous studies have demonstrated increased load of de novo copy number variants or single nucleotide variants in individuals with neurodevelopmental disorders, including epileptic encephalopathies, intellectual disability, and autism.

The Carboxyl Termini of KATP Channels Bind Nucleotides

ATP-sensitive potassium (KATP) channels are expressed in many excitable, as well as epithelial, cells and couple metabolic changes to modulation of cell activity. ATP regulation of KATP channel activity may involve direct binding of this nucleotide to the pore-forming inward rectifier (Kir) subunit despite the lack of known nucleotide-binding motifs. To examine this possibility, we assessed the binding of the fluorescent ATP analogue, 2′,3′-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)adenosine 5′-triphosphate (TNP-ATP) to maltose-binding fusion proteins of the NH2- and COOH-terminal cytosolic regions of the three known KATP channels (Kir1.1, Kir6.1, and Kir6.2) as well as to the COOH-terminal region of an ATP-insensitive inward rectifier K+ channel (Kir2.1). We show direct binding of TNP-ATP to the COOH termini of all three known KATP channels but not to the COOH terminus of the ATP-insensitive channel, Kir2.1. TNP-ATP binding was specific for the COOH termini of KATP channels because this nucleotide did not bind to the NH2 termini of Kir1.1 or Kir6.1. The affinities for TNP-ATP binding to KATP COOH termini of Kir1.1, Kir6.1, and Kir6.2 were similar. Binding was abolished by denaturing with 4 m urea or SDS and enhanced by reduction in pH. TNP-ATP to protein stoichiometries were similar for all KATP COOH-terminal proteins with 1 mol of TNP-ATP binding/mole of protein. Competition of TNP-ATP binding to the Kir1.1 COOH terminus by MgATP was complex with both Mg2+ and MgATP effects. Glutaraldehyde cross-linking demonstrated the multimerization potential of these COOH termini, suggesting that these cytosolic segments may directly interact in intact tetrameric channels. Thus, the COOH termini of KATPtetrameric channels contain the nucleotide-binding pockets of these metabolically regulated channels with four potential nucleotide-binding sites/channel tetramer.

Cardiac Potassium Channel Dysfunction in Sudden Infant Death Syndrome

Life-threatening arrhythmias have been suspected as one cause of the sudden infant death syndrome (SIDS), and this hypothesis is supported by the observation that mutations in arrhythmia susceptibility genes occur in 5-10% of cases. However, the functional consequences of cardiac potassium channel gene mutations associated with SIDS and how these alleles might mechanistically predispose to sudden death are unknown. To address these questions, we studied four missense KCNH2 (encoding HERG) variants, one compound KCNH2 genotype, and a missense KCNQ1 mutation all previously identified in Norwegian SIDS cases. Three of the six variants exhibited functional impairments while three were biophysically similar to wild-type channels (KCNH2 variants V279M, R885C, and S1040G). When co-expressed with WT-HERG, R273Q and K897T/R954C generated currents resembling the rapid component of the cardiac delayed rectifier current (I(Kr)) but with significantly diminished amplitude. Action potential modeling demonstrated that this level of functional impairment was sufficient to evoke increased action potential duration and pause-dependent early afterdepolarizations. By contrast, KCNQ1-I274V causes a gain-of-function in I(Ks) characterized by increased current density, faster activation, and slower deactivation leading to accumulation of instantaneous current upon repeated stimulation. Action potential simulations using a Markov model of heterozygous I274V-I(Ks) incorporated into the Luo-Rudy (LRd) ventricular cell model demonstrated marked rate-dependent shortening of action potential duration predicting a short QT phenotype. Our results indicate that certain potassium channel mutations associated with SIDS confer overt functional defects consistent with either LQTS or SQTS, and further emphasize the role of congenital arrhythmia susceptibility in this syndrome.

Strain- and age-dependent hippocampal neuron sodium currents correlate with epilepsy severity in Dravet syndrome mice.

Heterozygous loss-of-function SCN1A mutations cause Dravet syndrome, an epileptic encephalopathy of infancy that exhibits variable clinical severity. We utilized a heterozygous Scn1a knockout (Scn1a(+/-)) mouse model of Dravet syndrome to investigate the basis for phenotype variability. These animals exhibit strain-dependent seizure severity and survival. Scn1a(+/-) mice on strain 129S6/SvEvTac (129.Scn1a(+/-)) have no overt phenotype and normal survival compared with Scn1a(+/-) mice bred to C57BL/6J (F1.Scn1a(+/-)) that have severe epilepsy and premature lethality. We tested the hypothesis that strain differences in sodium current (INa) density in hippocampal neurons contribute to these divergent phenotypes. Whole-cell voltage-clamp recording was performed on acutely-dissociated hippocampal neurons from postnatal days 21-24 (P21-24) 129.Scn1a(+/-) or F1.Scn1a(+/-) mice and wild-type littermates. INa density was lower in GABAergic interneurons from F1.Scn1a(+/-) mice compared to wild-type littermates, while on the 129 strain there was no difference in GABAergic interneuron INa density between 129.Scn1a(+/-) mice and wild-type littermate controls. By contrast, INa density was elevated in pyramidal neurons from both 129.Scn1a(+/-) and F1.Scn1a(+/-) mice, and was correlated with more frequent spontaneous action potential firing in these neurons, as well as more sustained firing in F1.Scn1a(+/-) neurons. We also observed age-dependent differences in pyramidal neuron INa density between wild-type and Scn1a(+/-) animals. We conclude that preserved INa density in GABAergic interneurons contributes to the milder phenotype of 129.Scn1a(+/-) mice. Furthermore, elevated INa density in excitatory pyramidal neurons at P21-24 correlates with age-dependent onset of lethality in F1.Scn1a(+/-) mice. Our findings illustrate differences in hippocampal neurons that may underlie strain- and age-dependent phenotype severity in a Dravet syndrome mouse model, and emphasize a contribution of pyramidal neuron excitability.

P‐glycoprotein is not a swelling‐activated Cl− channel; possible role as a Cl− channel regulator

1 The whole‐cell configuration of the patch‐clamp technique was used to determine if P‐glycoprotein (Pgp) is a swelling‐activated Cl− channel. 2 Hamster pgp1 cDNA was transfected into a mouse fibroblast cell line resulting in expression of functional Pgp in the plasma membrane. This cell line was obtained without exposure to chemotherapeutic agents. 3 Swelling‐activated whole‐cell Cl− current (Icl,swell) was elicited by lowering the bath osmolality. Icl,swell, was characterized in detail in the pgp1‐transfected mouse cell line and compared with that of its parental cell line. Expression of Pgp did not modify the magnitude or properties of Icl,swell, except that addition of the anti‐Pgp antibody C219 to the pipette solution inhibited this current by 75% only in the Pgp‐expressing cells. 4 I Cl,swell in the mouse Pgp‐expressing cell line was compared with that in a Pgp‐expressing hamster fibroblast cell line. The characteristics of ICl,swell (voltage dependence, blocker sensitivity, anion selectivity sequence, requirement for hydrolysable ATP) in Pgp‐expressing cells were different between the two cell lines. These results suggest that the channel(s) responsible for ICl,swell are different between the two cell lines. In addition, C219 inhibited ICl,swell in both Pgp‐expressing cell lines, even though they seem to express different swelling‐activated Cl− channels. 5 We conclude that firstly, Pgp is not a swelling‐activated Cl− channel; secondly, it possibly functions as a Cl− channel regulator; and thirdly, ICl,swell is underlined by different Cl− channels in different cells.

Single-channel Properties of Human NaV1.1 and Mechanism of Channel Dysfunction in SCN1A-associated Epilepsy

Mutations in genes encoding neuronal voltage-gated sodium channel subunits have been linked to inherited forms of epilepsy. The majority of mutations (>100) associated with generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI) occur in SCN1A encoding the NaV1.1 neuronal sodium channel α-subunit. Previous studies demonstrated functional heterogeneity among mutant SCN1A channels, revealing a complex relationship between clinical and biophysical phenotypes. To further understand the mechanisms responsible for mutant SCN1A behavior, we performed a comprehensive analysis of the single-channel properties of heterologously expressed recombinant WT-SCN1A channels. Based on these data, we then determined the mechanisms for dysfunction of two GEFS+-associated mutations (R1648H, R1657C) both affecting the S4 segment of domain 4. WT-SCN1A has a slope conductance (17 pS) similar to channels found in native mammalian neurons. The mean open time is ∼0.3 ms in the −30 to −10 mV range. The R1648H mutant, previously shown to display persistent sodium current in whole-cell recordings, exhibited similar slope conductance but had an increased probability of late reopening and a subfraction of channels with prolonged open times. We did not observe bursting behavior and found no evidence for a gating mode shift to explain the increased persistent current caused by R1648H. Cells expressing R1657C exhibited conductance, open probability, mean open time, and latency to first opening similar to WT channels but reduced whole-cell current density, suggesting decreased number of functional channels at the plasma membrane. In summary, our findings define single-channel properties for WT-SCN1A, detail the functional phenotypes for two human epilepsy-associated sodium channel mutants, and clarify the mechanism for increased persistent sodium current induced by the R1648H allele.