ABCMdb

PMID: 8910333

Seibert FS, Linsdell P, Loo TW, Hanrahan JW, Riordan JR, Clarke DM
Cytoplasmic loop three of cystic fibrosis transmembrane conductance regulator contributes to regulation of chloride channel activity.
J Biol Chem. 1996 Nov 1;271(44):27493-9., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Ser945Leu ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg ABCC7 p.Gly970Arg ABCC7 p.His949Tyr ABCC7 p.His949Tyr Cytoplasmic Loop Three of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Regulation of Chloride Channel Activity* (Received for publication, May 3, 1996, and in revised form, August 22, 1996) Fabian S. Seibert‡, Paul Linsdell§¶, Tip W. Loo, John W. Hanrahan§ʈ, John R. Riordan**‡‡, and David M. Clarke§§ From the Medical Research Council Group in Membrane Biology, Departments of Medicine and Biochemistry, University of Toronto, Toronto, Ontario, Canada, M5S 1A8, the §Department of Physiology, McGill University, Montreal, Quebec, Canada, H3G 1Y6, and the **Mayo Graduate School of Medicine and Department of Biochemistry and Molecular Biology, S. C. Johnson Medical Research Center, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259 To examine the contribution of the large cytoplasmic loops of the cystic fibrosis transmembrane conductance regulator (CFTR) to channel activity, the three point-mutations (S945L, H949Y, G970R) were characterized that have been detected in the third cytoplasmic loop (CL3, residues 933-990) in patients with cystic fibrosis. Login to comment 1 ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg ABCC7 p.His949Tyr Chinese hamster ovary cell lines stably expressing wild-type 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. Login to comment 3 ABCC7 p.His949Tyr 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. Login to comment 5 ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg ABCC7 p.His949Tyr S945L-CFTR and G970R-CFTR showed a severe reduction in the P0, whereas the H949Y mutation doubled the P0 relative to wild-type. Login to comment 7 ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg 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. Login to comment 34 ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg ABCC7 p.His949Tyr The aim of the present study was to examine the functional significance of a previously uninvestigated domain, CL3 (predicted residues: 933-990, connecting TMs 8 and 9 of CFTR; Fig. 1), by characterizing the three different point-mutations that have been identified in CL3 from patients with CF (S945L (Claustres et al., 1993), H949Y (Ghanem et al., 1994), and G970R (Cuppens et al., 1993)). Login to comment 35 ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg ABCC7 p.His949Tyr The aim of the present study was to examine the functional significance of a previously uninvestigated domain, CL3 (predicted residues: 933-990, connecting TMs 8 and 9 of CFTR; Fig. 1), by characterizing the three different point-mutations that have been identified in CL3 from patients with CF (S945L (Claustres et al., 1993), H949Y (Ghanem et al., 1994), and G970R (Cuppens et al., 1993)). Login to comment 54 ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg ABCC7 p.His949Tyr Western blotting with the CFTR-specific monoclonal antibody M3A7 (Kartner et al., 1992) demonstrated that wild-type and G970R-mutant CFTRs yielded fully mature protein (170 kDa, band C) as the major product, whereas mutants S945L and H949Y yielded little of the mature form (Fig. 2). Login to comment 58 ABCC7 p.Ser945Leu ABCC7 p.His949Tyr These findings indicate that the S945L and H949Y mutations cause a defect in the biosynthetic processing pathways of CFTR maturation. Login to comment 62 ABCC7 p.Ser945Leu ABCC7 p.Ser945Leu In comparison to a control sample which remained at 37 °C without glycerol, it was observed that for the severely affected mutant S945L these procedures did not improve maturation of CFTR. Login to comment 63 ABCC7 p.His949Tyr In the case of H949Y, however, the amount of protein in band C was further increased under both conditions, with a more striking effect observed with 10% glycerol. Login to comment 68 ABCC7 p.Ser945Leu Very little stimulation occurred in cells expressing S945L-CFTR in agreement with the severe inhibition of protein maturation described above. Login to comment 69 ABCC7 p.His949Tyr H949Y-CFTR-containing cells exhibited iodide effluxes which were similar to wild-type, although there was much less mature protein than in wild-type expressing cells as judged by Western blotting. Login to comment 70 ABCC7 p.His949Tyr This suggested that the H949Y mutation may actually produce a hyperactive form of CFTR. Login to comment 71 ABCC7 p.Gly970Arg For G970R-CFTR, a forskolin-induced efflux was almost absent, despite the large amounts of complex glycosylated protein observed in Western blotting. Login to comment 79 ABCC7 p.Gly970Arg It was apparent that G970R allowed the production of fully glycosylated CFTR, but that there was little anion channel activity in the cells expressing this mutant. Login to comment 81 ABCC7 p.Gly970Arg Using this approach, it was observed that similar amounts of mature forms of G970R-CFTR and wild-type protein were biotinylated (Fig. 5). Login to comment 83 ABCC7 p.Gly970Arg This showed that G970R-CFTR did reach the cell surface and therefore must be severely impaired in function to explain the very low level of iodide efflux observed. Login to comment 84 ABCC7 p.Gly970Arg The absence of function is likely to be the cause of the CF symptoms in patients affected by the G970R mutation. Login to comment 88 ABCC7 p.Gly970Ala ABCC7 p.Gly970Met The G970A and G970M alterations resulted in efflux levels similar to wild-type, suggesting that amino acid size was not the major factor determining disturbance of CFTR function. Login to comment 89 ABCC7 p.Gly970Glu ABCC7 p.Gly970Lys Introducing a negative charge (G970E) decreased iodide efflux while the introduction of a positive charge (G970K) again abolished CFTR function. Login to comment 90 ABCC7 p.Gly970Arg Therefore, the loss of CFTR activity observed with the mutation G970R appears to be due to the presence of a positive charge at this site. Login to comment 116 ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg ABCC7 p.His949Tyr For each mutant, however, the level of channel activity was clearly different from wild-type CFTR (Fig. 7); both S945L and G970R channels showed a lower level of activity than wild-type, while for H949Y-CFTR activity appeared to be higher than wild-type. Login to comment 117 ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg ABCC7 p.His949Tyr This was confirmed by channel mean open probability (P0) measurements; S945L-CFTR and G970R-CFTR had significantly lower mean P0 values than wild-type channels, and the mean P0 of H949Y channels was significantly greater than observed for wild-type (Fig. 8A). Login to comment 118 ABCC7 p.Ser945Leu Analysis of channel burst kinetics indicated that for each mutant the alteration in P0 was mainly the result of a change in open burst duration (Fig. 8B), with a smaller change in interburst duration also contributing to the reduced P0 seen in S945L (Fig. 8C). Login to comment 122 ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg Both S945L and G970R mutants, however, had I-V relationships which were not completely linear over this voltage range, but instead showed slight outward rectification (Fig. 9, A and C). Login to comment 123 ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg In S945L-CFTR, this outward rectification was due to a significant reduction in current at negative membrane potentials compared to the wild-type channel (Fig. 9, A and D), while in G970R outward rectification resulted from increased current at positive potentials (Fig. 9, C and D). Login to comment 124 ABCC7 p.His949Tyr In contrast, H949Y channels had a linear I-V relationship (Fig. 9B) with a conductance similar to wild-type (7.9 Ϯ 0.1 pS; n ϭ 7). Login to comment 125 ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg The reasons for the outward rectification seen in both S945L and G970R are unclear; however, the fact that CL3 mutations can have subtle effects on channel conductance suggests that this region may be physically located close to the inner mouth of the CFTR chloride channel pore. Login to comment 128 ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg ABCC7 p.His949Tyr The most striking effect due to the amino acid substitutions was a drastically altered P0 of the mutant CFTRs relative to wild-type CFTR, with S945L and G970R decreasing the P0 of the channel and H949Y doubling its P0. Login to comment 131 ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg ABCC7 p.His949Tyr Within the framework of this model, the altered duration of the open state observed in the present study indicates that mutations in CL3 can affect events at NBF2 or affect communication from NBF2 to the pore. Mutations within CL3 can have opposite effects of either prolonging (H949Y) or dramatically decreasing (S945L, G970R) the duration of the open state. Login to comment 145 ABCC7 p.Ser945Leu ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg ABCC7 p.Gly970Arg tances due to mutations S945L and G970R show that mutagenesis of residues in CL3 has a subtle influence on pore properties, suggesting that CL3 may contribute to a region around the inner mouth of the pore. Login to comment 146 ABCC7 p.His949Tyr Notably, one of the CL3 mutations, H949Y, results in a channel which is more active than the one produced by nature. Login to comment 147 ABCC7 p.Pro574His A similar effect was noted previously for the CF-associated NBF1 mutation P574H. Login to comment 149 ABCC7 p.His949Tyr As seen for H949Y, this hyperactivity is mainly due to an increase in the mean open time of the channel, whereas conductance of the channel is not affected by the mutation. Login to comment 150 ABCC7 p.Pro574His ABCC7 p.His949Tyr The occurrence of substantial activity for these mutants correlates well with the observation that patients affected by the P574H and H949Y mutations suffer from a less severe form of CF and are pancreatic sufficient (Kerem et al., 1990; Ghanem et al., 1994). Login to comment 154 ABCC7 p.His949Tyr ABCC7 p.His949Tyr ABCC7 p.His949Tyr Specifically, the wild-type-like iodide efflux level together with the low expression level of H949Y-CFTR suggest a very high activity for that mutant, which cannot be fully accounted for by the doubling of the P0 observed in single-channel patch-clamping. However, iodide efflux may overestimate H949Y-CFTR activity if both wild-type CFTR expressing and H949Y-CFTR expressing cells reach an efflux plateau which masks a difference in the activity of their respective CFTR channels. Login to comment 161 ABCC7 p.Ser945Leu ABCC7 p.Gly970Arg ABCC7 p.His949Tyr Examples of wild-type, S945L, H949Y, and G970R CFTR channel currents recorded from inside-out patches at a membrane potential of -30 mV. Login to comment 168 ABCC7 p.Gly970Arg iodide efflux provides an efficient tool to identify gross alterations in CFTR channel activity, but the quantitative details are best evaluated by more subtle techniques such as single-channel patch-clamping. However, for low activity channels such as G970R-CFTR, which give little iodide efflux, the P0 may somewhat overestimate residual channel activity since only open channels can be observed electrophysiologically whereas channels which never open during the course of the experiment will be missed. Login to comment 174 ABCC7 p.Gly970Arg The significant decrease in CFTR function due to the G970R mutation is consistent with this point of view, since the loss of activity was due to the charge rather than size of the introduced Arg. Login to comment 175 ABCC7 p.Gly970Arg ABCC7 p.Gly970Arg This, and the observation that mature G970R-CFTR is properly trafficked to the plasma membrane, suggests that G970R affects activity through an altered electrostatic interaction within the protein or with other molecules rather than a gross change in CFTR structure. Login to comment 180 ABCC7 p.His949Tyr The only exception thus far is H949Y which already has some expression at the cell surface under normal growth conditions. Login to comment