ABCMdb

PMID: 8744306

Cheung M, Akabas MH
Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment.
Biophys J. 1996 Jun;70(6):2688-95., [PubMed]

Sentences

No. Mutations Sentence Comment

25 ABCC7 p.Lys335Glu A mutation, K335E, in M6 altered the permeability and conductance ratios for halides (Anderson et al., 1991b). Login to comment 26 ABCC7 p.Arg347His In the mutant R347H, multiple ion occupancy was dependent on the pH of the intracellular solution, and presumably titration of the histidine altered a nearby anion-binding site (Tabcharani et al., 1993). Login to comment 27 ABCC7 p.Arg347His The ability to titrate the histidine in the R347H mutant also suggests that Arg347 is on the water-exposed surface of the channel lining. Login to comment 85 ABCC7 p.Arg347Cys ABCC7 p.Thr339Cys The peak current at -100 mV was -7117 &#b1; 511 nA for the wild type, and ranged from -1709 &#b1; 124 nA for the R347C mutant to -7709 + 700 nA for the T339C mutant (Fig. 2 A). Login to comment 86 ABCC7 p.Arg347Cys ABCC7 p.Thr351Cys ABCC7 p.Thr339Cys The peak current at -100 mV was -7117 ± 511 nA for the wild type, and ranged from -1709 ± 124 nA for the R347C mutant to -7709 + 700 nA for the T339C mutant (Fig. 2 A). Login to comment 87 ABCC7 p.Thr351Cys The time for the currents to reach a steady-state level after application of the cAMP-activating solution was 20 + 1 min for wild type, and ranged from 14 + 2 min for 1344C to 51 + 4 min for T351C (Fig. 2 B). Login to comment 90 ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys ABCC7 p.Gln353Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Thr351Cys ABCC7 p.Met348Cys ABCC7 p.Leu333Cys ABCC7 p.Lys329Cys ABCC7 p.Val350Cys ABCC7 p.Ser341Cys ABCC7 p.Val345Cys ABCC7 p.Ala349Cys ABCC7 p.Phe337Cys ABCC7 p.Thr339Cys ABCC7 p.Leu346Cys Effects of MTS reagents on wild-type cysteines RESULTS in CFTR To identify the residues in and flanking the M6 membrane-spanning segment that are on the water-exposed surface of As reported previously (Akabas et al., 1994b), extracellular applications of the MTS reagents to Xenopus oocytes ex- L2j K329C L. _J *G330C 1331C 1332C L333C R334C K335C 1336C F337C T338C T339C 1340C S341C T342C C343,WT 1344C V345C L346C R347C M348C A349C V350C T351C R352C Q353C 0 2000 4000 6000 8000 0 25 50 PEAK CURRENTS (nA) TIME TO REACH PLATEAU (min) FIGURE 2 Peak CFTR-induced currents and time to reach the plateau current after stimulation with cAMP-activating reagents for 24 cysteine-substitution mutants and wild-type CFTR. Login to comment 91 ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys ABCC7 p.Gln353Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Thr351Cys ABCC7 p.Met348Cys ABCC7 p.Leu333Cys ABCC7 p.Lys329Cys ABCC7 p.Val350Cys ABCC7 p.Ser341Cys ABCC7 p.Val345Cys ABCC7 p.Ala349Cys ABCC7 p.Phe337Cys ABCC7 p.Thr339Cys ABCC7 p.Leu346Cys Effects of MTS reagents on wild-type cysteines RESULTS in CFTR To identify the residues in and flanking the M6 membrane-spanning segment that are on the water-exposed surface of As reported previously (Akabas et al., 1994b), extracellular applications of the MTS reagents to Xenopus oocytes ex- L2j K329C L. _J *G330C 1331C 1332C L333C R334C K335C 1336C F337C T338C T339C 1340C S341C T342C C343,WT 1344C V345C L346C R347C M348C A349C V350C T351C R352C Q353C 0 2000 4000 6000 8000 0 25 50 PEAK CURRENTS (nA) TIME TO REACH PLATEAU (min) FIGURE 2 Peak CFTR-induced currents and time to reach the plateau current after stimulation with cAMP-activating reagents for 24 cysteine-substitution mutants and wild-type CFTR. Login to comment 98 ABCC7 p.Arg347Cys .0 %0-0 0 10 20 30 40 50 MTSET' d]I 0-0 cAMP 0 0 0 10 20 30 40 50 Time (min) FIGURE 3 Effect of the MTS reagents on the CFTR-induced current in oocytes expressing the R347C mutant. Login to comment 99 ABCC7 p.Arg347Cys .0 %0-0 0 10 20 30 40 50 MTSET' d]I 0-0 cAMP 0 0 0 10 20 30 40 50 Time (min) FIGURE 3 Effect of the MTS reagents on the CFTR-induced current in oocytes expressing the R347C mutant. Login to comment 108 ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys ABCC7 p.Gln353Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr351Cys ABCC7 p.Leu333Cys ABCC7 p.Ser341Cys ABCC7 p.Phe337Cys Accessibility of substituted cysteines to MTSES- A 1-min application of 10 mM MTSES- significantly inhibited the CFIR-induced currents of 9 of the 24 cysteine-substituted mutants (Fig. 4 A), L333C, R334C, K335C, F337C, S341C, R347C, T351C, R352C, and Q353C. Login to comment 109 ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys ABCC7 p.Gln353Cys ABCC7 p.Gln353Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr351Cys ABCC7 p.Leu333Cys ABCC7 p.Ser341Cys ABCC7 p.Phe337Cys Accessibility of substituted cysteines to MTSES- A 1-min application of 10 mM MTSES- significantly inhibited the CFIR-induced currents of 9 of the 24 cysteine-substituted mutants (Fig. 4 A), L333C, R334C, K335C, F337C, S341C, R347C, T351C, R352C, and Q353C. Login to comment 110 ABCC7 p.Gln353Cys An 8-min application of 10mM MTSES- to these mutants did not markedly increase the inhibitory effects, except for the mutant Q353C, in which the extent of inhibition increased (Fig. 4 B); hence the reactions for the other mutants were complete within 1 min. Login to comment 113 ABCC7 p.Ile344Cys Another mutant, I344C, reacted with MTSEA+ more slowly, requiring an 8-min application of 2.5 mM MTSEA+ to significantly alter the current (Fig. 5 B). Login to comment 114 ABCC7 p.Ile344Cys Another mutant, I344C, reacted with MTSEA+ more slowly, requiring an 8-min application of 2.5 mM MTSEA+ to significantly alter the current (Fig. 5 B). Login to comment 115 ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys ABCC7 p.Arg334Cys We also examined the ability of a larger, permanently positively charged reagent, MTSET+, to react with three of the mutants, R334C, R347C, and R352C, that were susceptible to MTSEA+. Login to comment 116 ABCC7 p.Arg347Cys ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys We also examined the ability of a larger, permanently positively charged reagent, MTSET+, to react with three of the mutants, R334C, R347C, and R352C, that were susceptible to MTSEA+. Login to comment 117 ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys ABCC7 p.Arg334Cys One-minute and 8-min applications of 1 mM MTSET+ inhibited the CFTIR-mediated current of the mutants R334C by 53 ± 6% and 52 ± 7% (n = 3); R347C by 44 ± 2% and 36 + 3% (n = 3) (Fig. 3 D); and R352C by 46 ± 11% and 54.6 ± 10.5% (n = 3). Login to comment 133 ABCC7 p.Ile344Cys The slow rate of reaction of MTSEA' with the I344C mutant and the inability of the larger, anionic MTSES- to react with 1344C suggests that access of the reagents to this residue may be sterically limited, possibly by neighboring residues on other membrane-spanning segments. Login to comment 135 ABCC7 p.Ile344Cys The slow rate of reaction of MTSEA' with the I344C mutant and the inability of the larger, anionic MTSES- to react with 1344C suggests that access of the reagents to this residue may be sterically limited, possibly by neighboring residues on other membrane-spanning segments. Login to comment 160 ABCC7 p.Lys335Cys In this case the effects of modification on the K335C mutant by the MTS reagents would have to be indirect. Login to comment 162 ABCC7 p.Lys335Cys In this case the effects of modification on the K335C mutant by the MTS reagents would have to be indirect. Login to comment 171 ABCC7 p.Arg352Cys Diameter of the channel lumen The ability of MTSET+ to react with the R352C mutant indicates that it can penetrate from the extracellular end to this level. Login to comment 173 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys Diameter of the channel lumen The ability of MTSET+ to react with the R352C mutant indicates that it can penetrate from the extracellular end to this level. Login to comment 175 ABCC7 p.Arg352Cys Therefore, the channel diameter from the extracellular enid to the position of R352C, near the cytoplasmic end of the M6 segment, must be at least 6 A. Login to comment 188 ABCC7 p.Arg347His ABCC7 p.Arg347Asp The multiple ion occupancy effects were eliminated by mutation of Arg347 to Asp or His, and the single-channel conductance was reduced (Tabcharani et al., 1993). Login to comment 190 ABCC7 p.Arg347His ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp The multiple ion occupancy effects were eliminated by mutation of Arg347 to Asp or His, and the single-channel conductance was reduced (Tabcharani et al., 1993). Login to comment 191 ABCC7 p.Lys335Glu The mutation K335E, a water-accessible residue, altered the relative halide permeability and conductance sequences (Anderson et al., 199lb). Login to comment 192 ABCC7 p.Arg347Glu Curiously, the mutation R347E was reported to have little effect on the relative halide permeability or conductance sequences of the channel (Anderson et al., 1991b). Login to comment 193 ABCC7 p.Lys335Glu ABCC7 p.Ser341Ala The mutation K335E, a water-accessible residue, altered the relative halide permeability and conductance sequences (Anderson et al., 199lb). Login to comment 195 ABCC7 p.Ser341Ala Another mutation in M6, S341A, caused a fourfold reduction in the affinity for the voltage-dependent channel blocker diphenylamine-2-carboxylate (McDonough et al., 1994); we have shown that Ser341 is exposed in the channel lumen and thus could interact with a channel blocker. Login to comment 196 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro The mutations R334W and R347P have been shown to reduce single-channel conductance and open probability (Sheppard et al., 1993). Login to comment 198 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro The mutations R334W and R347P have been shown to reduce single-channel conductance and open probability (Sheppard et al., 1993). Login to comment