A mutation in the ␥ 2 subunit of the ␥-aminobutyric acid (GABA) type A receptor (GABAR), which changes an arginine to a glutamine at position 43 (R43Q), is linked to familial idiopathic epilepsies. We used radioligand binding, immunoblotting, and immunofluorescence techniques to examine the properties of wild-type ␣ 1 ␤ 2 ␥ 2 and mutant ␣ 1 ␤ 2 ␥ 2 R43Q GABARs expressed in HEK 293 cells. The ␥ 2 R43Q mutation had no affect on the binding affinity of the benzodiazepine flunitrazepam. However, in cells expressing ␣ 1 ␤ 2 ␥ 2 R43Q GABARs, the number of binding sites for [ 3 H]flunitrazepam relative to wild-type receptors was decreased 75%. Using surface protein biotinylation, affinity purification, and immunoblotting, we demonstrated that expression of cell surface ␣ 1 ␤ 2 ␥ 2 R43Q GABARs was decreased. Surface immunostaining of HEK 293 cells expressing ␣ 1 ␤ 2 ␥ 2 R43Q GABARs confirmed that surface expression of the ␥ 2 R43Q subunit was reduced. These data demonstrate that the ␥ 2 R43Q mutation impairs expression of cell surface GABARs. A deficit in surface GABAR expression would reduce synaptic inhibition and result in neuronal hyperexcitability, which could explain why families possessing the ␥ 2 R43Q subunit have epilepsy.Idiopathic partial and generalized epilepsies are the most common forms of heritable seizure disorders, accounting for 40% of all epilepsies (1). Idiopathic epilepsies are those seizure disorders that are not preceded by or concomitant with trauma or other disorders but are due mainly to genetic factors. Because of the ubiquitous role of the neurotransmitter GABA 1 in mediating cortical inhibition, deficits in GABAergic transmission have long been surmised to play a role in the pathogenesis of epilepsy. Recently, several mutations in the GABA A receptor (GABAR) have been identified through the use of genetic linkage analysis in families with idiopathic generalized epilepsies.GABARs are heteropentameric ligand gated chloride channels that mediate fast synaptic inhibition in the brain. The receptors are assembled from a number of different subunits and subunit isoforms, including ␣ 1-6 , ␤ 1-3 , ␥ 1-3 , ␦, ⑀, , and (2, 3). The majority of GABARs in the brain are believed to consist of 2 ␣, 2 ␤, and 1 ␥ subunits (4, 5). The inclusion of a ␥ subunit confers benzodiazepine (BZD) sensitivity to GABARs (6) and also influences receptor expression, synaptic targeting, and trafficking (7,8). To date, four mutations in the ␥ 2 subunit of the GABAR (missense mutations R43Q and K289M, the nonsense mutation Q351X, and the anomalous splice-donor site mutation IVS6 ϩ 2T 3 G) and one mutation in the ␣ 1 subunit of the receptor (missense mutation A322D) have been associated with idiopathic epileptic syndromes (9 -13).Several groups have explored the effects of the ␥ 2 R43Q mutation on GABAR function. Using recombinant GABAR expression in Xenopus laevis oocytes and two-electrode voltage clamp recording techniques, no differences in GABA EC 50 values between wild-type (WT)␣ 1 ␤ 2 ␥ 2 and mutant ␣ 1 ␤ 2 ␥ 2 R43Q r...

The large conductance, voltage- and calcium-dependent potassium (BK) channel serves as a major negative feedback regulator of calcium-mediated physiological processes and has been implicated in muscle dysfunction and neurological disorders. In addition to membrane depolarization, activation of the BK channel requires a rise in cytosolic calcium. Localization of the BK channel near calcium channels is therefore critical for its function. In a genetic screen designed to isolate novel regulators of the Caenorhabditis elegans BK channel, SLO-1, we identified ctn-1, which encodes an α-catulin homologue with homology to the cytoskeletal proteins α-catenin and vinculin. ctn-1 mutants resemble slo-1 loss-of-function mutants, as well as mutants with a compromised dystrophin complex. We determined that CTN-1 uses two distinct mechanisms to localize SLO-1 in muscles and neurons. In muscles, CTN-1 utilizes the dystrophin complex to localize SLO-1 channels near L-type calcium channels. In neurons, CTN-1 is involved in localizing SLO-1 to a specific domain independent of the dystrophin complex. Our results demonstrate that CTN-1 ensures the localization of SLO-1 within calcium nanodomains, thereby playing a crucial role in muscles and neurons.

Plasmid vectors used for mammalian expression or for in vitro cRNA translation can differ substantially and are rarely cross-compatible. To make comparisons between mammalian and Xenopus oocyte expression systems, it would be advantageous to use a single vector without the need for shuttle vectors or subcloning. We have designed such a vector, designated pUNIV for universal, with elements that will allow for in vitro or ex vivo expression in multiple cell types. We tested the expression of pUNIV-based cDNA cassettes using enhanced green fluorescent protein and two forms of the type A γ-aminobutyric acid receptor (GABA A R) and compared pUNIV to vectors optimized for expression in either Xenopus oocytes or mammalian cells. In HEK293 cells, radioligand binding was robust, and patch clamp experiments showed that subtle macroscopic GABA A R kinetics were indistinguishable from our previous results. In Xenopus oocytes, agonist median effective concentration measurements matched previous work using a vector optimized for oocyte expression. Furthermore, we found that expression using pUNIV was significantly enhanced in oocytes and was remarkably long-lasting in both systems.

The imidazopyridine zolpidem (Ambien)

No abstract