The cystic fibrosis gene product (CFTR) is a complex protein that functions as an adenosine 3,5-monophosphate (cAMP)-stimulated ion channel and possibly as a regulator of intracellular processes. In order to determine whether the CFTR molecule contains a functional aqueous pathway, anion, water, and urea transport were measured in Xenopus oocytes expressing CFTR. Cyclic AMP agonists induced a Cl- conductance of 94 microsiemens and an increase in water permeability of 4 x 10(-4) centimeter per second that was inhibited by a Cl- channel blocker and was dependent on anion composition. CFTR has a calculated single channel water conductance of 9 x 10(-13) cubic centimeter per second, suggesting a pore-like aqueous pathway. Oocytes expressing CFTR also showed cAMP-stimulated transport of urea but not the larger solute sucrose. Thus CFTR contains a cAMP-stimulated aqueous pore that can transport anions, water, and small solutes. The results also provide functional evidence for water movement through an ion channel.

Abstract. CHIP28 is a 28-kD hydrophobic integral membrane protein that functions as a water channel in erythrocytes and renal tubule epithelial cell membranes. We examined the transmembrane topology of CHIP28 in the ER by engineering a reporter of translocation (derived from bovine prolactin) into nine sequential sites in the CHIP28 coding region. The resulting chimeras were expressed in Xenopus oocytes, and the topology of the reporter with respect to the ER membrane was determined by protease sensitivity. We found that although hydropathy analysis predicted up to seven potential transmembrane regions, CHIP28 spanned the membrane only four times. Two putative transmembrane helices, residues 52-68 and 143-157, reside on the lumenal and cytosolic surfaces of the ER membrane, respectively. Topology derived from these chimeric proteins was supported by cell-free translation of five truncated CHIP28 cDNAs, by N-linked glycosylation at an engineered consensus site in native CHIP28 (residue His69), and by epitope tagging of the CHIP28 amino terminus. Defined protein chimeras were used to identify internal sequences that direct events of CHIP28 topogenesis. A signal sequence located within the first 52 residues initiated nascent chain translocation into the ER lumen. A stop transfer sequence located in the hydrophobic region from residues 90-120 terminated ongoing translocation. A second internal signal sequence, residues 155-186, reinitiated translocation of a COOH-terminal domain (residues 186-210) into the ER lumen. Integration of the nascent chain into the ER membrane occurred after synthesis of 107 residues and required the presence of two membrane-spanning regions. From this data, we propose a structural model for CHIP28 at the ER membrane in which four membrane-spanning a-helices form a central aqueous channel through the lipid bilayer and create a pathway for water transport,

The expression of receptors for cholecystokinin (CCK) and other similar acting Ca2 -mobilizing hormones was studied in Xenopus laevis oocytes. Poly(A)+ RNA was prepared from pancreatic AR42J cells, which normally express receptors for CCK and bombesin and the RNA injected into oocytes. The presence of these pancreatic receptors on the oocytes was then demonstrated by hormone-induced mobilization of &5Ca2 CCK receptors were present 1 day (maxnum, 2 days) after injection of RNA and were generally proportional to the amount of poly(A)+ RNA injected (1-50 ng Cholecystokinin (CCK) is a gastrointestinal hormone that acts to regulate secretion of the exocrine and endocrine pancreas, gallbladder contraction, and gastric emptying (1,2). Moreover, it is also present in brain and other neural tissue where it appears to function as a neurotransmitter or neuromodulator (3-6). The actions of CCK on peripheral target cells are mediated by enhanced inositol phospholipid turnover, diacylglycerol formation, and mobilization of intracellular Ca2" (7). These actions are initiated by specific membrane receptors, which have been characterized as to number, specificity, size, and subunit composition (8)(9)(10)(11)(12)

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