Epilepsy affects more than 0.5 % of the population in the world. Genetic factors play an important role in many of the idiopathic generalized epilepsies (IGEs) and in some partial epilepsies. GABAA receptors (GABARs) are the major inhibitory receptors in the CNS, and mutations in ?2, d and al GABAR genes are associated with IGEs. To understand the bases for IGEs associated with GABAR mutations, it is necessary to determine the functional, assembly and trafficking errors produced by the mutations. Many of these are single nucleotide missense mutations, but recently mutations that introduce a premature translation- termination codon (PTC) and mutations in splice donor sites have been reported. PTCs might produce nonsense mediated decay (NMD) of mRNA if the mutation is not in the last exon or produce a truncated subunit if it is in the last exon. Splice donor site mutations produce mutant protein by: (1) exon skipping, (2) use of cryptic splice site within the down stream intron, or (3) intron inclusion if the intron is small. It is possible all three mechanisms would generate a PTC, thus triggering NMD. The goals of this proposal are to characterize the altered expression, function and trafficking produced by ?2 epilepsy PTC and splice donor site mutations. Hypotheses are that the: a) ?2S(Q351X) PTC mutation reduces het al?2?2S(Q351X) current by producing a C-terminal truncated subunit that assembles to form mutant receptors that reduce trafficking of wt receptors, are co trafficked with wt receptors to the cell surface and have altered function, b) ?2S(Q1X) PTC mutation reduces het al?2?2L(Q1X) receptor current by triggering NMD of mutant mRNA, thus producing haploinsufficiency. c) ?2S (IVS6+2T-G) splice donor site mutation reduces het al?2?2L (IVS6+2T-G) receptor current via haploinsufficiency by triggering NMD through either exon 6 skipping, resulting in a PTC at the joining site of exon 5 and exon 7, or by using intron 6 downstream cryptic splice sites, also resulting in a PTC, or by generating a truncated protein due to incomplete NMD (NMD inefficiency).
Specific aims are to determine the pathophysiological alterations in translation, trafficking, surface expression and pharmacological and biophysical properties of het and horn a) al?2?2 (Q351X), b) al?2?2 (Q1X), and c) al?2?2 (IVS6+2T-G) receptors expressed in fibroblasts and ?2S siRNA treated cultured hippocampal neurons.
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