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A missense mutation of the Na+ channel alpha II subunit gene Na(v)1.2 in a patient with febrile and afebrile seizures causes channel dysfunction.

ABSTRACT: Generalized epilepsy with febrile seizures plus (GEFS+), a clinical subset of febrile seizures (FS), is characterized by frequent episodes beyond 6 years of age (FS+) and various types of subsequent epilepsy. Mutations in beta1 and alpha(I)-subunit genes of voltage-gated Na(+) channels have been associated with GEFS+1 and 2, respectively. Here, we report a mutation resulting in an amino acid exchange (R188W) [corrected] in the gene encoding the alpha-subunit of neuronal voltage-gated Na(+) channel type II (Na(v)1.2) in a patient with FS associated with afebrile seizures. The mutation R188W [corrected] occurring on Arg(187), a highly conserved residue among voltage-gated Na(+) channels, was not found in 224 alleles of unaffected individuals. Whole-cell patch clamp recordings on human embryonic kidney (HEK) cells expressing a rat wild-type (rNa(v)1.2) and the corresponding mutant channels showed that the mutant channel inactivated more slowly than wild-type whereas the Na(+) channel conductance was not affected. Prolonged residence in the open state of the R188W [corrected] mutant channel may augment Na(+) influx and thereby underlie the neuronal hyperexcitability that induces seizure activity. Even though a small pedigree could not show clear cosegregation with the disease phenotype, these findings strongly suggest the involvement of Na(v)1.2 in a human disease and propose the R188W [corrected] mutation as the genetic defect responsible for febrile seizures associated with afebrile seizures.

SUBMITTER: Sugawara T

PROVIDER: S-EPMC33477 | biostudies-literature | 2001 May

REPOSITORIES: biostudies-literature

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A missense mutation of the Na+ channel alpha II subunit gene Na(v)1.2 in a patient with febrile and afebrile seizures causes channel dysfunction.

Sugawara T T Tsurubuchi Y Y Agarwala K L KL Ito M M Fukuma G G Mazaki-Miyazaki E E Nagafuji H H Noda M M Imoto K K Wada K K Mitsudome A A Kaneko S S Montal M M Nagata K K Hirose S S Yamakawa K K

Proceedings of the National Academy of Sciences of the United States of America 20010501 11

Generalized epilepsy with febrile seizures plus (GEFS+), a clinical subset of febrile seizures (FS), is characterized by frequent episodes beyond 6 years of age (FS+) and various types of subsequent epilepsy. Mutations in beta1 and alpha(I)-subunit genes of voltage-gated Na(+) channels have been associated with GEFS+1 and 2, respectively. Here, we report a mutation resulting in an amino acid exchange (R188W) [corrected] in the gene encoding the alpha-subunit of neuronal voltage-gated Na(+) chann ...[more]

PMID: 11371648

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Project description:See Lerche (doi:10.1093/brain/awy073) for a scientific commentary on this article.Proline-rich transmembrane protein 2 (PRRT2) is the causative gene for a heterogeneous group of familial paroxysmal neurological disorders that include seizures with onset in the first year of life (benign familial infantile seizures), paroxysmal kinesigenic dyskinesia or a combination of both. Most of the PRRT2 mutations are loss-of-function leading to haploinsufficiency and 80% of the patients carry the same frameshift mutation (c.649dupC; p.Arg217Profs*8), which leads to a premature stop codon. To model the disease and dissect the physiological role of PRRT2, we studied the phenotype of neurons differentiated from induced pluripotent stem cells from previously described heterozygous and homozygous siblings carrying the c.649dupC mutation. Single-cell patch-clamp experiments on induced pluripotent stem cell-derived neurons from homozygous patients showed increased Na+ currents that were fully rescued by expression of wild-type PRRT2. Closely similar electrophysiological features were observed in primary neurons obtained from the recently characterized PRRT2 knockout mouse. This phenotype was associated with an increased length of the axon initial segment and with markedly augmented spontaneous and evoked firing and bursting activities evaluated, at the network level, by multi-electrode array electrophysiology. Using HEK-293 cells stably expressing Nav channel subtypes, we demonstrated that the expression of PRRT2 decreases the membrane exposure and Na+ current of Nav1.2/Nav1.6, but not Nav1.1, channels. Moreover, PRRT2 directly interacted with Nav1.2/Nav1.6 channels and induced a negative shift in the voltage-dependence of inactivation and a slow-down in the recovery from inactivation. In addition, by co-immunoprecipitation assays, we showed that the PRRT2-Nav interaction also occurs in brain tissue. The study demonstrates that the lack of PRRT2 leads to a hyperactivity of voltage-dependent Na+ channels in homozygous PRRT2 knockout human and mouse neurons and that, in addition to the reported synaptic functions, PRRT2 is an important negative modulator of Nav1.2 and Nav1.6 channels. Given the predominant paroxysmal character of PRRT2-linked diseases, the disturbance in cellular excitability by lack of negative modulation of Na+ channels appears as the key pathogenetic mechanism.

Project description:P/Q-type channels are the principal presynaptic calcium channels in brain functioning in neurotransmitter release. They are composed of the pore-forming CaV2.1 ?1 subunit and the auxiliary ?2?-2 and ?4 subunits. ?4 is encoded by CACNB4, and its multiple splice variants serve isoform-specific functions as channel subunits and transcriptional regulators in the nucleus. In two siblings with intellectual disability, psychomotor retardation, blindness, epilepsy, movement disorder and cerebellar atrophy we identified rare homozygous variants in the genes LTBP1, EMILIN1, CACNB4, MINAR1, DHX38 and MYO15 by whole-exome sequencing. In silico tools, animal model, clinical, and genetic data suggest the p.(Leu126Pro) CACNB4 variant to be likely pathogenic. To investigate the functional consequences of the CACNB4 variant, we introduced the corresponding mutation L125P into rat ?4b cDNA. Heterologously expressed wild-type ?4b associated with GFP-CaV1.2 and accumulated in presynaptic boutons of cultured hippocampal neurons. In contrast, the ?4b-L125P mutant failed to incorporate into calcium channel complexes and to cluster presynaptically. When co-expressed with CaV2.1 in tsA201 cells, ?4b and ?4b-L125P augmented the calcium current amplitudes, however, ?4b-L125P failed to stably complex with ?1 subunits. These results indicate that p.Leu125Pro disrupts the stable association of ?4b with native calcium channel complexes, whereas membrane incorporation, modulation of current density and activation properties of heterologously expressed channels remained intact. Wildtype ?4b was specifically targeted to the nuclei of quiescent excitatory cells. Importantly, the p.Leu125Pro mutation abolished nuclear targeting of ?4b in cultured myotubes and hippocampal neurons. While binding of ?4b to the known interaction partner PPP2R5D (B56?) was not affected by the mutation, complex formation between ?4b-L125P and the neuronal TRAF2 and NCK interacting kinase (TNIK) seemed to be disturbed. In summary, our data suggest that the homozygous CACNB4 p.(Leu126Pro) variant underlies the severe neurological phenotype in the two siblings, most likely by impairing both channel and non-channel functions of ?4b.

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Dataset Information

A missense mutation of the Na+ channel alpha II subunit gene Na(v)1.2 in a patient with febrile and afebrile seizures causes channel dysfunction.

Publications

A missense mutation of the Na+ channel alpha II subunit gene Na(v)1.2 in a patient with febrile and afebrile seizures causes channel dysfunction.

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