ABCMdb

PMID: 9305991

Seibert FS, Jia Y, Mathews CJ, Hanrahan JW, Riordan JR, Loo TW, Clarke DM
Disease-associated mutations in cytoplasmic loops 1 and 2 of cystic fibrosis transmembrane conductance regulator impede processing or opening of the channel.
Biochemistry. 1997 Sep 30;36(39):11966-74., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Gly178Arg ABCC7 p.Glu193Lys 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. Login to comment 4 ABCC7 p.Glu193Lys 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. Login to comment 7 ABCC7 p.Ile148Thr ABCC7 p.Gly178Arg 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. Login to comment 106 ABCC7 p.Ile148Thr ABCC7 p.Arg297Gln ABCC7 p.Gly178Arg ABCC7 p.Glu193Lys ABCC7 p.Ile175Val However, only the mutations I148T, I175V, G178R, E193K, and R297Q allowed wild-type-like maturation of the protein to the fully glycosylated 170 kDa species (band C). Login to comment 107 ABCC7 p.Gly149Arg ABCC7 p.Arg258Gly ABCC7 p.Asp192Gly ABCC7 p.His139Arg The remaining amino acid substitutions significantly decreased the yield of band C, with relative amounts of "vector only" (background) < G149R-CFTR < H139R-CFTR < R258G-CFTR < D192G-CFTR , wild-type CFTR (Figure 2, bottom). Login to comment 113 ABCC7 p.His949Tyr The treatment did not promote processing to a degree detectable by Western blotting (Figure 3); this finding was not unexpected because of all mutations in the CLs examined thus far, only the H949Y-CFTR variant could be rescued to some degree (18, 19, 21; unpublished observations). Login to comment 120 ABCC7 p.Gly149Arg ABCC7 p.Ile148Thr ABCC7 p.Arg258Gly ABCC7 p.Arg297Gln ABCC7 p.Asp192Gly ABCC7 p.His139Arg ABCC7 p.Ile175Val In accordance with reduced levels of processing, the H139R, G149R, D192G, and R258G mutations significantly decreased the anion translocation capability of CFTR, whereas the properly processed I148T, I175V, and R297Q variants allowed iodide movement comparable to that of wild type. Login to comment 121 ABCC7 p.Gly178Arg ABCC7 p.Gly178Arg ABCC7 p.Glu193Lys The only exceptions to this scheme were the G178R and E193K variants, which both produced activities that were lower than predicted from their wild-type-like maturation profile; in all five experiments analyzed, the decrease was more severe for G178R-CFTR-expressing cells. Login to comment 124 ABCC7 p.Gly178Arg ABCC7 p.Glu193Lys Still, to ensure that the decreased levels of activity of G178R-CFTR- and E193K-CFTR-expressing cells were not the result of a post-ER targeting defect but that the CFTR variants indeed reached their site of action, surface labeling was performed with the membrane-impermeant reagent biotin-LC-hydrazide (4042). Login to comment 128 ABCC7 p.Gly178Arg ABCC7 p.Glu193Lys To further characterize decreases in the anion permeation profile of G178R-CFTR and E193K-CFTR and to observe potentially small changes in the chloride channel activity of the remaining three maturation-competent mutants, the more sensitive patch-clamping method was applied. Login to comment 131 ABCC7 p.Glu193Lys A striking exception was E193K that, in agreement with the iodide efflux data for intact cells, produced a significant decrease in the open probability (Po) of CFTR when measured in excised patches (Figure 7A). Login to comment 133 ABCC7 p.Glu193Lys Interestingly, however, the reduction in Po was due to an increase in the mean closed time of the E193K channels (Figure 7C), in marked contrast to the effect of CL3 mutations, which modified the mean open time of CFTR (21). Login to comment 146 ABCC7 p.Gly178Arg For G178R-CFTR, which had greatly reduced anion translocation capability in the iodide efflux assay, alterations in single-channel kinetics could not be identified by the patch-clamp technique. Login to comment 147 ABCC7 p.Ile148Thr ABCC7 p.Gly178Arg This discrepancy may originate from the additional observation that G178R-CFTR (as well as I148T-CFTR) could not be locked open by the nonhydrolyzable ATP analogue AMP-PNP. Login to comment 149 ABCC7 p.Ile148Thr ABCC7 p.Gly178Arg Thus, the number of channels per patch may have been underestimated for I148T-CFTR- and G178R-CFTR-expressing cells, resulting in a systematic overestimation of the Po. Login to comment 153 ABCC7 p.Gly149Arg ABCC7 p.Arg258Gly ABCC7 p.Asp192Gly ABCC7 p.His139Arg When reconstructed in heterologous expression systems, four of the amino acid substitutions (H139R, G149R, D192G, and R258G) inhibited maturation and transport of CFTR to the cell surface, so that the protein cannot carry out its regular functions at that location. Login to comment 154 ABCC7 p.Gly178Arg ABCC7 p.Glu193Lys Two additional mutations, G178R and E193K, significantly reduced CFTR`s anion translocation capability as observed by iodide efflux assays. Login to comment 155 ABCC7 p.Glu193Lys In the case of the E193K variants the reduced iodide efflux was explained by the decreased Po found in single-channel patches. Login to comment 171 ABCC7 p.Gly178Arg ABCC7 p.Glu193Lys The finding that the two mutations in CLs 1 and 2 with the most severe effects on the chloride channel activity of CFTR introduce a positive charge into CL1 (G178R and E193K) is consistent with an important role of electrostatic interactions in the normal functioning of the loops. Login to comment 175 ABCC7 p.Glu193Lys In contrast, the decreased anion translocation capability caused by mutations in CL1, in the case of E193K, resulted from an increase in the mean closed time. Login to comment 187 ABCC7 p.Gly178Arg Note that some charge changes did allow full maturation, e.g., G178R. Login to comment 190 ABCC7 p.Ile148Thr ABCC7 p.Arg297Gln ABCC7 p.Ile175Val I148T, I175V, and R297Q did not adversely affect the processing, gating, or conductance of CFTR. Login to comment 199 ABCC7 p.Ile148Thr ABCC7 p.Arg297Gln ABCC7 p.Ile175Val The entire CFTR gene was not sequenced in patients with mutations I148T, I175V, or R297Q when these mutations were published. Login to comment 203 ABCC7 p.Gly178Arg ABCC7 p.Glu193Lys Mutations in CLs 1 and 3 had drastic effects on the ability of CFTR to respond to regulatory stimuli: E193K in CL1 decreased the opening rate, in agreement with the decreased Po of a CL1 deletion variant (19) and the reduced iodide efflux activity of G178R-CFTR, whereas mutations in CL3 affected the duration of the open state (21). Login to comment 204 ABCC7 p.Arg297Gln In contrast, R297Q, the only CF-associated mutation that could be evaluated in CL2 or deletion of CL2 (18), apparently had little effect on the chloride channel activity of CFTR, as was shown previously for mutations in CL4 (20, 22). Login to comment 208 ABCC7 p.Ile148Thr (B) Mean single-channel current-voltage relationship of wild-type CFTR and I148T-CFTR as a representative CL 1 and 2 mutant. Login to comment 209 ABCC7 p.Ile148Thr Each point represents the mean ( SE (where this is larger than the size of the symbol) of data from four (I148T) or five (WT) patches. Login to comment 217 ABCC7 p.Ile148Thr ABCC7 p.Gly178Arg Note that the I148T- and G178R-CFTR variants could not be locked open with AMP-PNP, so that for these mutants the number of channels in each patch may have been underestimated. Login to comment