Glycerol Reverses the Misfolding Phenotype of the Most Common Cystic Fibrosis Mutation

doi:10.1074/jbc.271.2.635

Glycerol Reverses the Misfolding Phenotype of the Most Common Cystic Fibrosis Mutation (*)

From the (1)Department of Biological Sciences and
(2)Department of Psychology, Cystic Fibrosis Research Laboratory, Stanford University, Stanford, California 94305-5020

^¶ To whom correspondence should be addressed. Tel.: 415-723-7581; Fax: 415-723-8475; kopito{at}leland.stanford.edu.

Abstract

The common ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) interferes with the biosynthetic folding of nascent CFTR polypeptides, leading to their retention and rapid degradation in an intracellular compartment proximal to the Golgi apparatus. Neither the pathway by which wild-type CFTR folds nor the mechanism by which the Phe deletion interferes with this process is well understood. We have investigated the effect of glycerol, a polyhydric alcohol known to stabilize protein conformation, on the folding of CFTR and ΔF508 in vivo. Incubation of transient and stable ΔF508 tranfectants with 10% glycerol induced a significant accumulation of ΔF508 protein bearing complex N-linked oligosaccharides, indicative of their transit to a compartment distal to the endoplasmic reticulum (ER). This accumulation was accompanied by an increase in mean whole cell cAMP activated chloride conductance, suggesting that the glycerol-rescued ΔF508 polypeptides form functional plasma membrane CFTR channels. These effects were dose- and time-dependent and fully reversible. Glycerol treatment also stabilized immature (core-glycosylated) ΔF508 and CFTR molecules that are normally degraded rapidly. These effects of glycerol were not due to a general disruption of ER quality control processes but appeared to correlate with the degree of temperature sensitivity of specific CFTR mutations. These data suggest a model in which glycerol serves to stabilize an otherwise unstable intermediate in CFTR biosynthesis, maintaining it in a conformation that is competent for folding and subsequent release from the ER quality control apparatus.

Footnotes

↵^§ Supported by the Cystic Fibrosis Foundation and a Human Frontiers Science Program long term fellowship.
↵* This work was supported by National Institutes of Health Grant DK43994. This work was done during the tenure of an established investigatorship of the American Heart Association (to R. R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

CF

cystic fibrosis

CFTR

cystic fibrosis transmembrane conductance regulator

DMEM

Dulbecco's modified Eagle's medium

FCS

fetal calf serum

ER

endoplasmic reticulum.
- Received October 23, 1995.
- Revision received November 15, 1995.