Disease-associated Mutations in the Fourth Cytoplasmic Loop of Cystic Fibrosis Transmembrane Conductance Regulator Compromise Biosynthetic Processing and Chloride Channel Activity

Abstract

A cluster of 18 point mutations in exon 17b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been detected in patients with cystic fibrosis. These mutations cause single amino acid substitutions in the most C-terminal cytoplasmic loop (CL4, residues 1035-1102) of the CFTR chloride channel. Heterologous expression of the mutants showed that 12 produced only core-glycosylated CFTR, which was retained in the endoplasmic reticulum; the other six mutants matured and reached the cell surface. In some cases substitution of one member of pairs of adjacent residues resulted in misprocessing, whereas the other did not. Thus, the secondary structure of CL4 may contribute crucially to the proper folding of the entire CFTR molecule. Cyclic AMP-stimulated iodide efflux was not detected from cells expressing the misprocessed variants but was from the other six, indicating that their mutations cause relatively subtle channel defects. Consistent with this, these latter mutations generally are present in patients who are pancreatic-sufficient, while the processing mutants are mostly from patients who are pancreatic-insufficient. Single-channel patch-clamp analysis demonstrated that the processed mutants had the same ohmic conductance as wild-type CFTR, but a lower open probability, generally due to an increase in channel mean closed time and a reduction in mean open time. This suggests that mutations in CL4 do not affect pore properties of CFTR, but disrupt the mechanism of channel gating.

Cytoplasmic Loop Three of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Regulation of Chloride Channel Activity

“…These results, together with those of Xie et al (1995) and Seibert et al (1996), attribute a significant role to the CLs in CFTR function. The observed effects are different for the various loops; CL2 is found to influence open channel properties by participating in the regulation of different subconductance states (Xie et al, 1995).…”

“…Construction, Expression, and Detection of CL3 Mutants-Mutagenesis was performed in the expression vectors pNUT-CFTR and pcDNA3-CFTR as described (Tabcharani et al, 1991;Seibert et al, 1996). The sequence of each polymerase chain reaction fragment was verified after insertion into the vector using the T7 Sequencing Kit (Pharmacia Biotech Inc.).…”

Cysteine Substitutions Reveal Dual Functions of the Amino-terminal Tail in Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating

“…CFTR channel gating probably involves physical and functional interactions among the R domain and the NBDs (7,9,10), although the precise roles of these domains in controlling channel opening and closing are still unclear. In addition, there have been reports that other regions of CFTR such as the cytoplasmic loops may also modulate CFTR channel function (11,12). We observed previously (13,14) that a cluster of negatively charged residues in a putative helical region of the aminoterminal tail (N-tail) participates in CFTR channel gating.…”

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.…”