Receptors that couple to 2 classes of G proteins increase cAMP and activate CFTR expressed in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl- channel activated by phosphorylation, was expressed in Xenopus oocytes along with various combinations of several other components of the cAMP signalling pathway. Activation of the coexpressed beta 2 adrenergic receptor increased cAMP and led to CFTR activation. The activation of CFTR (1) requires only short (15 s) exposure to isoproterenol, (2) occurs for agonist concentrations 100-1000 fold lower than those that produce cAMP increases detectable by a radioimmunoassay, (3) requires injection of only 5 pg of receptor cRNA per oocyte, and (4) can be increased further by coexpression of cRNA for adenylyl cyclase type II or III or for Gs alpha. In addition, CFTR activation and cAMP increases by beta 2 activation were enhanced by activation of the coexpressed 5HT1A receptor, which is thought to couple to Gi. The additional activation by the 5HT1A receptor was enhanced by coexpression of adenylyl cyclase type II but not with type III and may proceed via the beta gamma subunits of a G protein. The sensitivity of the assay system is also demonstrated by responses to vasoactive intestinal peptide and to pituitary adenylate cyclase-activating polypeptide in oocytes injected with cerebral cortex mRNA.

The molecular biology of chloride channels.

The molecular biology of chloride channels is a new field, having begun only 5 years ago with the cloning, on the basis of partial amino acid sequence information, of complementary DNAs for subunits of the neuronal glycine and gamma-aminobutyric acid-receptor channels. The sequences of these subunits that form heterooligomeric channels revealed a general similarity with the structural motif employed by the well-studied and previously cloned nicotinic acetylcholine-receptor subunits. The principal distinguishing feature was the rings of positively charged residues within the loops separating the putative transmembrane helices of the new chloride selective channels. A great diversity of subunit types with specific functional and pharmacologic properties as well as localizations within the central nervous system have been identified and cloned. Chloride channels are known to play an important role in both secretion and reabsorption of salt and fluid across wet epithelia, and the cystic fibrosis transmembrane conductance regulator, cloned by genetic means, has been shown to be centrally involved in both of these processes. It is regulated by a complex mechanism involving both ATP binding and phosphorylation at multiple sites. Expression cloning techniques have yielded a voltage-gated family of channels with a large number of potential membrane spanning segments; one member is responsible for the resting potential of skeletal muscle. An entirely different low molecular weight-voltage-gated channel, possibly regulated by osmolarity changes, is the most recent chloride channel to be cloned. It almost certainly will not be the last.

Plasma membrane lipids of human diploid fibroblasts from normal individuals and patients with cystic fibrosis.

The lipid composition of isolated plasma membranes of human skin fibroblasts is described for the first time. Plasma membranes from a number of strains of fibroblasts from patients with cystic fibrosis and matched normals were isolated by a recently described procedure and analysed for major phospholipid classes, cholesterol and fatty acids. No differences in the quantities of these compounds were detected between cells of the two different origins. The fetal calf serum used to supplement the growth medium contained relatively more palmitoleate and oleate but less stearate than the membranes. There were also no consistent differences between cystic fibrosis and normal membranes in terms of the fatty acid compositions of their individual phospholipid classes. Consistent with this lack of chemical change in the lipids of membranes of cystic fibrosis cells, the degree of fluorescence polarization of diphenylhexatriene, an index of fluidity, was also unchanged.