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

Abstract

To examine the contribution of the large cytoplasmic loops of the cystic fibrosis transmembrane conductance regulator (CFTR) to channel activity, the three pointmutations (S945L, H949Y, G970R) were characterized that have been detected in the third cytoplasmic loop (CL3, residues 933-990) in patients with cystic fibrosis. Chinese hamster ovary cell lines stably expressing wildtype CFTR or mutant G970R-CFTR yielded polypeptides with apparent masses of 170 kDa as the major products, whereas the major products of mutants S945L-CFTR and H949Y-CFTR had apparent masses of 150 kDa. The 150-kDa forms of CFTR were sensitive to endoglycosidase H digestion, indicating that these mutations interfered with maturation of the protein. Increased levels of mature CFTR (170 kDa) could be obtained for mutant H949Y when cells were grown at a lower temperature (26°C) or incubated in the presence of 10% glycerol. For all mutants, the open probability (P 0 ) of the CFTR channels was significantly altered. S945L-CFTR and G970R-CFTR showed a severe reduction in the P 0 , whereas the H949Y mutation doubled the P 0 relative to wild-type. The changes in P 0 predominantly resulted from an alteration of the mean burst durations which suggests that CL3 is involved in obtaining and/or maintaining stability of the open state. In addition, mutants S945L and G970R had current-voltage relationships that were not completely linear over the range ؎80 mV, but showed slight outward rectification. The fact that CL3 mutations can have subtle effects on channel conductance indicates that this region may be physically close to the inner mouth of the pore.

Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3

“…However, S945L and H949Y, found in patients with CF, are close to Lys-946 and His-950 of CL3 and stop maturation of the protein (31). What is more, CL3 also involves ubiquitinationdependent CFTR trafficking (32).…”

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.…”

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).…”