Febrile seizures (FS) and temporal lobe epilepsy (TLE) were found in four of the seven siblings born to healthy Moroccan consanguineous parents. We hypothesized autosomal recessive (AR) inheritance. Combined linkage analysis and autozygosity mapping of a genome-wide single nucleotide polymorphism genotyping identified a unique identical by descent (IBD) locus of 9.6 Mb on human chromosome 8q12.1-q13.2. Sequencing of the 38 genes mapped within the linked interval revealed a homozygous missense mutation c.809C>T (p.Ala270Val) in the carboxypeptidase A6 gene (CPA6). Screening all exons of CPA6 in unrelated patients with partial epilepsy (n = 195) and FS (n = 145) revealed a new heterozygous missense mutation c.799G>A (p.Gly267Arg) in three TLE patients. Structural modeling of CPA6 indicated that both mutations are located near the enzyme's active site. In contrast to wild-type CPA6, which is secreted and binds to the extracellular matrix where it is enzymatically active, Ala270Val CPA6 was secreted at about 40% of the level of the wild-type CPA6 and was fully active, while Gly267Arg CPA6 was not detected in the medium or extracellular matrix. This study suggests that CPA6 is genetically linked to an AR familial form of FS and TLE, and is associated with sporadic TLE cases.

We have studied the earliest expression of GABA-induced CI- channels in the rat embryonic dorsal spinal cord (DSC) using in situ hybridization, immunocytochemistry, flow cytometry and electrophysiology. At embryonic day 13 (E13) cells in the dorsal region are still proliferating. In situ hybridization consistently showed transcripts encoding only three GABAA receptor subunits (alpha4, beta1 and gammal); immunocytochemistry both in tissue sections and in acutely isolated cells in suspension demonstrated the expression of the corresponding proteins and also revealed staining for other subunits (alpha2, alpha3, beta3, gamma2). In patch-recordings performed in cells acutely isolated from the dorsal cord, responses to GABA were detected in 356 out of 889 cells. GABA-evoked responses, which often displayed the opening of a few channels, were mediated by CI- ions, were inhibited by bicuculline and picrotoxin, and potentiated by benzodiazepines. Taken together, these observations indicate that CI- channels likely involve GABAA type receptors. Fluctuation analysis revealed channel kinetics consisting of three exponential components (Ts: approximately 1,9 and 90 ms) and a wide variety of inferred unitary conductance values, ranging between 4 and 40 pS. A comparison of these results with observations in other, later embryonic cell types and recombinant receptors suggests that most of the earliest E13 DSC GABAA receptors may include alpha3 subunit. These GABAA receptor Cl- channels may be activated physiologically as both GABA synthesizing enzymes and GABA are present in the E13 dorsal cord.

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