Two conserved arginines in the extracellular N-terminal domain of the GABA(A) receptor alpha(5) subunit are crucial for receptor function.

The gamma-aminobutyric acid (GABA) binding pocket within the GABA(A) receptor complex has been suggested to contain arginine residues. The aim of this study was to test this hypothesis by mutating arginine residues potentially contributing to the formation of a GABA binding pocket. Thus, six arginines conserved in human GABA(A) receptor alpha subunits (arginine 34, 70, 77, 123, 135, and 224) as well as two nonconserved arginines (79 and 190), all located in the extracellular N-terminal segment of the alpha(5) subunit, were substituted by lysines. The individual alpha(5) subunit mutants were coexpressed with human beta(2) and gamma(2s) GABA(A) receptor subunits in Chinese hamster ovary cells by transient transfection. Electrophysiological whole-cell patch-clamp recordings show that, of the eight arginine residues tested, the two arginines at positions 70 and 123 appear to be essential for the GABA-gated chloride current because the EC(50) values of the two mutant constructs increase >100-fold compared with the wild-type alpha(5),beta(2), gamma(2s) GABA(A) receptor. However, diazepam and allopregnanolone modulation and pentobarbital stimulation properties are unaffected by the introduction of lysines at positions 70 and 123. A double mutant carrying lysine substitutions at positions 70 and 123 is virtually insensitive to GABA, suggesting alterations of one or more GABA binding sites.

Identification and cloning of a gamma 3 subunit splice variant of the human GABA(A) receptor.

cDNA sequences encoding two forms of the GABA(A) gamma 3 receptor subunit were cloned from human hippocampus. The nucleotide sequences differ by the absence (gamma 3S) or presence (gamma 3L) of 18 bp located in the presumed intracellular loop between transmembrane region (TM) III and IV. The extra 18 bp in the gamma 3L subunit generates a consensus site for phosphorylation by protein kinase C (PKC). Analysis of human genomic DNA encoding the gamma 3 subunit reveals that the 18 bp insert is contiguous with the upstream proximal exon.

Intrinsic termination of T7 RNA polymerase mediated by either RNA or DNA.

Intrinsic termination of T7 RNA polymerase transcription occurs at different signals in vitro. One type of signal is similar to that mediating factor-independent termination of Escherichia coli RNA polymerase, whereas the other type does not involve RNA hairpin formation. By examining the termination behaviour of T7 RNA polymerase at the E.coli rrnB operon t1 terminator, at the T7-t(phi) terminator, at the human preproparathyroid hormone gene terminator on both single- and double-stranded templates, and in the presence of GTP or ITP during transcription, we show that the termination event can be mediated by either RNA or DNA structural features. Moreover, by using co-transcriptional probing with potassium permanganate, we present evidence for the presence of transcription-induced hyperreactive thymidines on the non-template strand in the DNA-mediated event, and a putative sequence motif is identified. We conclude that intrinsic termination of T7 RNA polymerase transcription in vitro can be mediated either by a hairpin in the nascent RNA or by a sequence motif including hyperreactive thymidines in the non-template DNA strand.