Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7 Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients

https://science.sciencemag.org/content/sci/245/4922/1066.full.pdf

Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.

This chapter discusses the gamma-aminobutyric acid (GABA) receptor channels, which are the most abundant inhibitory neurotransmitter in the CNS. Following release from presynaptic vesicles, GABA exerts fast inhibitory effects by interacting with GABA receptors, whose primary function is to hyperpolarize neuronal membranes in mature CNS neurons. GABA receptors are found both presynaptically, where they decrease the likelihood of neurotransmitter release, and postsynaptically, where they decrease the likelihood of neuronal firing. There are two types of GABA receptor, termed GABA A and GABA B receptors. GABA A receptors are fast-activating Clˉ channels from the Cys-loop family of ligand-gated ion channels. Activation of GABA A receptors causes membrane hyperpolarization by allowing Clˉ influx, reflecting the relatively low concentration of Clˉ found intracellularly in most adult CNS neurons. GABA A receptors can also mediate depolarizing responses in most immature CNS neurons and in mature peripheral neurons.

GABAA Receptor Channels

Four cloned cDNAs encoding 900-amino acid putative glutamate receptors with approximately 70 percent sequence identity were isolated from a rat brain cDNA library. In situ hybridization revealed differential expression patterns of the cognate mRNAs throughout the brain. Functional expression of the cDNAs in cultured mammalian cells generated receptors displaying alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective binding pharmacology (AMPA = quisqualate greater than glutamate greater than kainate) as well as cation channels gated by glutamate, AMPA, and kainate and blocked by 6,7-dinitroquinoxaline-2,3-dione (CNQX).

https://science.sciencemag.org/content/sci/249/4968/556.full.pdf

A family of AMPA-selective glutamate receptors

Receptors for N-methyl-D-aspartate (NMDA) are involved in many plastic and pathological processes in the brain. Glycine has been reported to potentiate NMDA responses in neurons and in Xenopus oocytes injected with rat brain messenger RNA. Glycine is now shown to be absolutely required for activation of NMDA receptors in oocytes. In voltage-clamped oocytes, neither perfusion nor rapid pressure application of NMDA onto messenger RNA-injected oocytes caused a distinct ionic current without added glycine. When glycine was added, however, NMDA evoked large inward currents. The concentration of glycine required to produce a half-maximal response was 670 nanomolar, and the glycine dose-response curve extrapolated to zero in the absence of glycine. Several analogs of glycine could substitute for glycine, among which D-serine and D-alanine were the most effective. The observation that D-amino acids are effective will be important in developing drugs targeted at the glycine site.

http://science.sciencemag.org/content/sci/241/4867/835.full.pdf

Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes

NEUROTRANSMISSION effected by GABA (γ-aminobutyric acid) is predominantly mediated by a gated chloride channel intrinsic to the GABAA receptor. This heterooligomeric receptor1 exists in most inhibitory synapses in the vertebrate central nervous system (CNS) and can be regulated by clinically important compounds such as benzodiazepines and barbiturates2. The primary structures of GABAA receptor α- and β-subunits have been deduced from cloned complementary DNAs3,4. Co-expression of these subunits in heterologous systems generates receptors which display much of the pharmacology of their neural counterparts, including potentiation by barbiturates3–5. Conspicuously, however, they lack binding sites for, and consistent electrophysiological responses to, benzodiazepines4,5. We now report the isolation of a cloned cDNA encoding a new GABAA receptor subunit, termed γ2, which shares approximately 40% sequence identity with α-and β-subunits and whose messenger RNA is prominently localized in neuronal subpopulations throughout the CNS. Importantly, coexpression of the γ2 subunit with α1 and β1 subunits produces GABAA receptors displaying high-affinity binding for central benzodiazepine receptor ligands.

Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology.

Amino-acid sequences derived from complementary DMAs encoding the α- and β-subunits of the GAB A/ benzo diazepine receptor from bovine brain show homology with other ligand-gated receptor subunits, suggesting that there is a super-family of ion-channel-containing receptors. Co-expression of the in vitro-generated α-subunit and β-subunit RNAs in Xenopus oocytes produces a functional receptor and ion channel with the pharmacological properties characteristic of the GABAA receptor.

David R. Burt

Papers

Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family