title: Pure haploinsufficiency for Dravet syndrome Na(V)1.1 (SCN1A) sodium channel truncating mutations.
abstract: OBJECTIVE
  Dravet syndrome (DS), a devastating epileptic encephalopathy, is mostly caused by mutations of the SCN1A gene, coding for the voltage-gated Na(+) channel Na(V)1.1 α subunit. About 50% of SCN1A DS mutations truncate Na(V)1.1, possibly causing complete loss of its function. However, it has not been investigated yet if Na(V)1.1 truncated mutants are dominant negative, if they impair expression or function of wild-type channels, as it has been shown for truncated mutants of other proteins (e.g., Ca(V) channels). We studied the effect of two DS truncated Na(V)1.1 mutants, R222* and R1234*, on coexpressed wild-type Na(+) channels.
  
  
  METHODS
  We engineered R222* or R1234* in the human cDNA of Na(V)1.1 (hNa(V)1.1) and studied their effect on coexpressed wild-type hNa(V)1.1, hNa(V)1.2 or hNa(V)1.3 cotransfecting tsA-201 cells, and on hNa(V)1.6 transfecting an human embryonic kidney (HEK) cell line stably expressing this channel. We also studied hippocampal neurons dissociated from Na(V)1.1 knockout (KO) mice, an animal model of DS expressing a truncated Na(V)1.1 channel.
  
  
  RESULTS
  We found no modifications of current amplitude coexpressing the truncated mutants with hNa(V)1.1, hNa(V)1.2, or hNa(V)1.3, but a 30% reduction coexpressing them with hNa(V)1.6. However, we showed that also coexpression of functional full-length hNa(V)1.1 caused a similar reduction. Therefore, this effect should not be involved in the pathomechanism of DS. Some gating properties of hNa(V)1.1, hNa(V)1.3, and hNa(V)1.6 were modified, but recordings of hippocampal neurons dissociated from Na(V)1.1 KO mice did not show any significant modifications of these properties. Therefore, Na(V)1.1 truncated mutants are not dominant negative, consistent with haploinsufficiency as the cause of DS.
  
  
  CONCLUSIONS
  We have better clarified the pathomechanism of DS, pointed out an important difference between pathogenic truncated Ca(V)2.1 mutants and hNa(V)1.1 ones, and shown that hNa(V)1.6 expression can be reduced in physiologic conditions by coexpression of hNa(V)1.1. Moreover, our data may provide useful information for the development of therapeutic approaches.
authors.affiliation: Department of Neurophysiopathology, Besta Foundation Neurological Institute, Milan, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Bechi
authors.forename: Paolo
authors.initials: P
authors.lastname: Scalmani
authors.forename: Emanuele
authors.initials: E
authors.lastname: Schiavon
authors.forename: Raffaella
authors.initials: R
authors.lastname: Rusconi
authors.forename: Silvana
authors.initials: S
authors.lastname: Franceschetti
authors.forename: Massimo
authors.initials: M
authors.lastname: Mantegazza
mesh_terms: D000818:Animals; D002460:Cell Line; D004594:Electrophysiology; D004831:Epilepsies, Myoclonic / Q000235:genetics*; D057809:HEK293 Cells; D057895:Haploinsufficiency; D006624:Hippocampus / Q000166:cytology / Q000502:physiology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D016296:Mutagenesis; D062550:NAV1.1 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins / Q000172:deficiency / Q000235:genetics*; D009474:Neurons / Q000502:physiology*; D018408:Patch-Clamp Techniques; D010957:Plasmids; D015222:Sodium Channels / Q000172:deficiency / Q000235:genetics*; D013577:Syndrome; D014162:Transfection
chemical_list: D062550:NAV1.1 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins; C568242:SCN1A protein, human; C568249:Scn1a protein, mouse; D015222:Sodium Channels