Journal
JOURNAL OF NEUROSCIENCE
Volume 24, Issue 24, Pages 5570-5578Publisher
SOC NEUROSCIENCE
DOI: 10.1523/JNEUROSCI.1301-04.2004
Keywords
juvenile myoclonic epilepsy; chloride ion channel; GABA(A) receptors; mutant; myoclonus; patch clamp; protein; concatamer
Categories
Funding
- NINDS NIH HHS [R01 NS033300, K08 NS044257, NS33300, NS39479, K08 NS44257-01, R01 NS039479] Funding Source: Medline
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Individuals with autosomal dominant juvenile myoclonic epilepsy are heterozygous for a GABA(A) receptor alpha1 subunit mutation (alpha1A322D). GABA(A) receptor alphabetagamma subunits are arranged around the pore in a beta-alpha-beta-alpha-gamma sequence (counterclockwise from the synaptic cleft). Therefore, each alpha1 subunit has different adjacent subunits, and heterozygous expression of alpha1(A322D), beta, and gamma subunits could produce receptors with four different subunit arrangements: beta-alpha1-beta-alpha1-gamma (wild type); beta-alpha1(A322D)-beta-alpha1-gamma (Het(betaalphabeta)); beta-alpha1-beta- alpha1(A322D)-gamma(Het(betaalphagamma)); beta-alpha1(A322D)-beta-alpha1(A322D)-gamma(homozygous). Expression of a 1: 1 mixture of wild-type and alpha1(A322D) subunits with beta2S and gamma2S subunits ( heterozygous transfection) produced smaller currents than wild type and much larger currents than homozygous mutant transfections. Western blot and biotinylation assays demonstrated that the amount of total and surface alpha1 subunit from heterozygous transfections was also intermediate between those of wild-type and homozygous mutant transfections. alpha1( A322D) mutations were then made in covalently tethered triplet (gamma2S-beta2S-alpha1) and tandem (beta2S-alpha1) concatamers to target selectively alpha1( A322D) to each of the asymmetric alpha1 subunits. Coexpression of mutant and wild-type concatamers resulted in expression of either Het(betaalphabeta) or Het(betaalphagamma) receptors. Het(betaalphabeta) currents were smaller than wild type and much larger than Het(betaalphagamma) and homozygous currents. Furthermore, Het(betaalphabeta) transfections contained less beta-alpha concatamer than wild type but more than both Het(betaalphagamma) and homozygous mutant transfections. Thus, whole-cell currents and protein expression of heterozygous alpha1(A322D) beta2Sgamma2S receptors depended on the position of the mutant alpha1 subunit, and GABA(A) receptor currents in heterozygous individuals likely result primarily from wild-type and Het(betaalphabeta) receptors with little contribution from Het(betaalphagamma) and homozygous receptors.
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