Disease-Associated Mutations in Cytoplasmic Loops 1 and 2 of Cystic Fibrosis Transmembrane Conductance Regulator Impede Processing or Opening of the Channel

Abstract

Since little is known about the contribution to function of the N-terminal cytoplasmic loops (CL1, residues 139-194; CL2, residues 242-307) of cystic fibrosis transmembrane conductance regulator (CFTR), all nine point mutations identified in CLs 1 and 2 from patients with cystic fibrosis were reconstructed in the expression vector pcDNA3-CFTR and expressed transiently in COS-1 and HEK-293 cells and stably in Chinese hamster ovary (CHO) cells. Four amino acid substitutions retarded production of mature, fully glycosylated CFTR, suggesting that misprocessing of the channel causes the disease symptoms in the affected patients. Protein maturation could not be promoted by cell culture conditions of reduced temperature (26 degrees C). When properly processed mutants were evaluated for functional defects by the iodide efflux method, the G178R- and E193K-CFTR-expressing cell lines showed impaired anion translocation activities. Patch-clamp studies of single channels revealed that E193K variants had a significantly decreased open probability, which resulted from an increase in the mean closed time of the channels. This contrasted with a previous study of disease-associated point mutations in CL3 that mainly affected the mean open time. None of the maturation-competent CL 1 and 2 mutants had altered conductance. Thus, the N-terminal CLs appear not to contribute to the anion translocation pathway of CFTR; rather, mutations in CL1 can impede transition to the open state. Interestingly, the ability of the non-hydrolyzable ATP analogue adenylyl imidodiphosphate (AMP-PNP) to lock the channel into open bursts was abolished by the I148T and G178R amino acid substitutions.

Human P-glycoprotein Contains a Greasy Ball-and-Socket Joint at the Second Transmission Interface

“…Detailed analysis of the effect of IH2 mutations in CFTR have not yet been performed. Preliminary studies on the effects of ICL1 and ICL2 mutations in CFTR that cause cystic fibrosis have shown that the ICL1 mutations have more inhibitory effects on CFTR function (40).…”

Mutation-Specific Potency and Efficacy of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Potentiators

“…Mutations lying outside the NBDs, in particular, in the CFTR intracellular loops (ICL1-ICL4), which connect the transmembrane segments, also cause a gating defect (Seibert et al, 1996(Seibert et al, , 1997. ICLs now are considered particularly interesting because they may couple changes in NBD conformation to movements of transmembrane segments, thus allowing gating of the CFTR pore (Mendoza and Thomas, 2007;He et al, 2008;Mornon et al, 2008;Serohijos et al, 2008).…”

Assessing the Disease-Liability of Mutations in CFTR

“…Mutations that have a frequency in CF patients exceeding 1% have been investigated in a variety of expression systems for their effect on the splicing of CFTR RNA or the function of the CFTR protein (Cheng et al 1990;Gregory et al 1991;Sheppard et al 1993). Several dozen other mutations reported in patients have been investigated for their functional effects (e.g., Seibert et al 1996aSeibert et al , 1997. The functional consequences of the remaining 1850 CFTR mutations are unknown.…”