ABCMdb

PMID: 7511062

Sheppard DN, Ostedgaard LS, Rich DP, Welsh MJ
The amino-terminal portion of CFTR forms a regulated Cl- channel.
Cell. 1994 Mar 25;76(6):1091-8., [PubMed]

Sentences

No. Mutations Sentence Comment

17 ABCC7 p.Asp836* To test whether the amino-terminal portion of CFfR could form a regulated Cl- channel, we constructed the CFTR mutant D836X. Login to comment 20 ABCC7 p.Asp836* We used two antibodies: M13-1 against the R domain, which is present in D636X, and M24-1 against the carboxyl terminus, which is absent in D836X. Login to comment 30 ABCC7 p.Asp836* Under basal conditions, whole-cell currents from cells expressing D836X had a relatively linear current-voltage (I-V) relationship and a reversal potential consistent with Cl--selectivity, whereas basal whole-cell currents from cells expressing wild-type CFTR were much less Clse- lective (Table 1). Login to comment 32 ABCC7 p.Asp836* The anion permeability and conductance sequences of D836X whole-cell currents were similar to those of wild-type CFTR (Br > Cl- > I-; Table 1). Login to comment 42 ABCC7 p.Asp836* After excision of membrane patches from cells expressing D836X, we observed no channel activity. Login to comment 45 ABCC7 p.Asp836* Phosphorylation with PKA (75 nM) significantly increased the P, of D836X channels. Login to comment 48 ABCC7 p.Asp836* These data suggest that some aspects of the relationship between the R domain and the rest of the channel may be altered in D836X, while others may remain intact. Login to comment 51 ABCC7 p.Asp836* The single-channel slope conductance of D836X (8.03 * 0.23 pS; n = 8) was not different from that of wild-type CFTR (8.29 -c 0.15 pS; n = 4). Login to comment 54 ABCC7 p.Asp836* Consistent with this interpretation is the finding that CAMP-activated whole-cell currents were observed in 10 of 59 cells expressing D836X (17%) compared with 10 of 14 cells expressing wild-type CFTR (71%). Login to comment 58 ABCC7 p.Asp836* Because D836X lacks NBDZ, we speculated that the interaction with intracellular nucleotides might be altered. Login to comment 63 ABCC7 p.Asp836* As we previously reported for wild-type CFTR (Anderson and Welsh, 1992), an Eadie-Hofstee plot of the D836X data generated a curved line(Figure 3C). Login to comment 67 ABCC7 p.Asp836* Figures 4A and 48 show that intracellular ADP (1 mM) produced equivalent reductions in the activity of both D836X and wild-type channels. Login to comment 68 ABCC7 p.Asp836* D636X Sediments as a Multlmer on Sucrose Gradient Centrlfugatlon Based on these results and considerations discussed below, we asked whether D836X might exist as a multimer. Login to comment 71 ABCC7 p.Asp836* 1094 A ATP D836X ...... PKA + ATP Is CFTR ATP _..... PKA + ATP _._... B 0.6 0.4 PO 0.2 0 0.6 0.4 PO 0.2 0 I CFTR \TP PKA + ATP ATP PKA + ATP Figure 2. Login to comment 81 ABCC7 p.Asp836* These sedimentation patterns A D836X C ~PAL 1s 0.4 PO 0.2 0 :;::::` 0 pl&~P] (2) 4.0 c k 5 2.0 n" 0 -_i 0 0.40.2 PO Figure 3. Login to comment 95 ABCC7 p.Asp836* However, our previous data suggest that full-length CFTR does not associate to form a multimer ;h&mino-Terminal Half of CFTR Forms a Cl- Channel A D836X ATP CFTR B ---- - IS 1s Figure 4. Login to comment 96 ABCC7 p.Asp836* Intracellular ADP Inhibits D&36X Cl- Channels (A) Comparison of the effect of ADP on the activity of three D836X Cl- channels and a single wild-type Cl- channel following PKAdependent phosphorylation. Login to comment 98 ABCC7 p.Asp836* ATP (0.88 mM MgATP) and ADP (1 mM) were added to the intracellular solution as indicated. Voltage was -86 mV (D836X) and -83 mV (CFTR), respectively. Login to comment 101 ABCC7 p.Asp836* Voltage was -86 f 1 mV (D836X) and -85 * 2 mV (CFTR). Login to comment 124 ABCC7 p.Asp836* The Membrane-Spanning Domains The conductive properties of Cl- channels formed by D636X were remarkably similar to those of wild-type CFTR: the channels had the same anion-to-cation permeability, anion selectivity sequence, single-channel conduc- 1096 A kDa 210 170 116 94 76 67 i B kDa 210 170 116 94 76 57 D836X 6% MO% Total % Sucrow Lysate CFTR % Sucrose ~0% Total Lysate 246 kDa 9/oSucrose - 20% Figure 5. Login to comment 141 ABCC7 p.Asp836* We found that although large amountsof protein were produced in cellsexpressing D836X, the number of functional Cl- channels generated was much less than that observed with wild-type CFfR. Login to comment 142 ABCC7 p.Asp836* Thus, D836X was not as efficient as wild type at generating functional channels. Login to comment 145 ABCC7 p.Trp846* Implications for Cystic Fibrosis A number of CF-associated nonsense mutations have been reported that would be predicted to generate a protein truncated after the R domain; examples include W846X and R851Xvidaud et al., 1996; Whiteet al., 1991). Login to comment 149 ABCC7 p.Asp836* Could a CF-associated mutant that made protein with properties similar to those of D836X produce sufficient residual activity to confer a milder clinical phenotype? Login to comment 150 ABCC7 p.Asp836* Given the limited expression of functional D836X Cl- channels despite relatively high-level protein expression, such a possibility seems unlikely. Login to comment 244 ABCC7 p.Arg553* ABCC7 p.Trp1316* Severe deficiency of cystic fibrosis transmembrane conductance regulator messenger RNA carrying nonsense mutations R553X and W1316X in respiratory epithelial cells of patients with cystic fibrosis. Login to comment