PMID: 22160394

Cui G, Song B, Turki HW, McCarty NA
Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers.
Pflugers Arch. 2012 Mar;463(3):405-18. Epub 2011 Dec 13., [PubMed]
Sentences
No. Mutations Sentence Comment
57 ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:57:76
status: NEW
view ABCC7 p.Arg334Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:57:85
status: NEW
view ABCC7 p.Arg334Ala details
For simplification, names of mutant channels are often abbreviated (e.g., R334A is R334A-CFTR). Login to comment
119 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:119:112
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:119:105
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:119:98
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:119:91
status: NEW
view ABCC7 p.Arg334Ala details
ABCC7 p.Ile344Ala
X
ABCC7 p.Ile344Ala 22160394:119:119
status: NEW
view ABCC7 p.Ile344Ala details
ABCC7 p.Arg347Ala
X
ABCC7 p.Arg347Ala 22160394:119:126
status: NEW
view ABCC7 p.Arg347Ala details
ABCC7 p.Arg352Ala
X
ABCC7 p.Arg352Ala 22160394:119:151
status: NEW
view ABCC7 p.Arg352Ala details
ABCC7 p.Met348Ala
X
ABCC7 p.Met348Ala 22160394:119:133
status: NEW
view ABCC7 p.Met348Ala details
ABCC7 p.Val350Ala
X
ABCC7 p.Val350Ala 22160394:119:140
status: NEW
view ABCC7 p.Val350Ala details
The major effects of increasing or decreasing sensitivity to Glyb were seen with mutations R334A, K335A, F337A, S341A, I344A, R347A, M348A, V350A, and R352A (Fig. 3 left). Login to comment
120 ABCC7 p.Ile344Ala
X
ABCC7 p.Ile344Ala 22160394:120:149
status: NEW
view ABCC7 p.Ile344Ala details
ABCC7 p.Val345Ala
X
ABCC7 p.Val345Ala 22160394:120:170
status: NEW
view ABCC7 p.Val345Ala details
The results were nearly identical for block by Glip (Fig. 3 middle) with one important distinction that significant effects were seen with mutations I344A (for Glyb) and V345A (for Glip), suggesting that these two highly related molecules share binding sites. Login to comment
125 ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:125:20
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:125:10
status: NEW
view ABCC7 p.Arg334Ala details
Mutations R334A and K335A lie in the outer vestibule of the pore of CFTR; surprisingly, the two mutations db ca 2 nA 100 ms 100 ms 2 nA Concentration (μM) 0 200 400 600 800 1000 0.2 0.4 0.6 0.8 1.0 Fractionalblock 100 ms 4 nA Fig. 2 Concentration-dependent block of WT-CFTR by Glyb, Glip and Tolb. Login to comment
133 ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:133:27
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:133:0
status: NEW
view ABCC7 p.Arg334Ala details
R334A weakened block while K335A strengthened block by both blockers. Login to comment
135 ABCC7 p.Arg334Cys
X
ABCC7 p.Arg334Cys 22160394:135:33
status: NEW
view ABCC7 p.Arg334Cys details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:135:23
status: NEW
view ABCC7 p.Arg334Ala details
Also, we reported that R334A and R334C exhibited multiple single-channel conductance levels, including subconductance 1 (s1), subconductance 2 (s2), and full conductance states (f). Login to comment
136 ABCC7 p.Arg334Cys
X
ABCC7 p.Arg334Cys 22160394:136:0
status: NEW
view ABCC7 p.Arg334Cys details
R334C can be modified by methanethiosulfonate reagents in a state-dependent manner suggesting that its position moves during channel gating [46]. Login to comment
137 ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:137:17
status: NEW
view ABCC7 p.Lys335Ala details
On the contrary, K335A single-channel behavior is very similar to that of WT-CFTR except the single-channel conductance is slightly decreased (-0.56± 0.01 pA, n=5; see Fig. 9), as would be expected for an amino acid that affects attraction of Cl- ions. Login to comment
140 ABCC7 p.Arg347Ala
X
ABCC7 p.Arg347Ala 22160394:140:10
status: NEW
view ABCC7 p.Arg347Ala details
ABCC7 p.Arg352Ala
X
ABCC7 p.Arg352Ala 22160394:140:20
status: NEW
view ABCC7 p.Arg352Ala details
Mutations R347A and R352A also represent a separate category from the rest. Login to comment
144 ABCC7 p.Asp993Arg
X
ABCC7 p.Asp993Arg 22160394:144:97
status: NEW
view ABCC7 p.Asp993Arg details
ABCC7 p.Arg352Glu
X
ABCC7 p.Arg352Glu 22160394:144:91
status: NEW
view ABCC7 p.Arg352Glu details
These functional characteristics were returned to approximately their wildtype behavior in R352E/D993R-CFTR, perhaps because the salt bridge was retained in this second-site reversion [12]. Login to comment
145 ABCC7 p.Arg347Ala
X
ABCC7 p.Arg347Ala 22160394:145:37
status: NEW
view ABCC7 p.Arg347Ala details
ABCC7 p.Arg352Ala
X
ABCC7 p.Arg352Ala 22160394:145:47
status: NEW
view ABCC7 p.Arg352Ala details
The present data show that mutations R347A and R352A significantly reduced block by all three blockers; for Glyb and Glip, block became strictly time-independent, perhaps reflecting the gross loss of pore architecture leading to loss of the binding site underlying slow pore block. Login to comment
150 ABCC7 p.Asn1138Ala
X
ABCC7 p.Asn1138Ala 22160394:150:48
status: NEW
view ABCC7 p.Asn1138Ala details
ABCC7 p.Thr1142Ala
X
ABCC7 p.Thr1142Ala 22160394:150:64
status: NEW
view ABCC7 p.Thr1142Ala details
ABCC7 p.Ser1149Ala
X
ABCC7 p.Ser1149Ala 22160394:150:88
status: NEW
view ABCC7 p.Ser1149Ala details
ABCC7 p.Val1147Ala
X
ABCC7 p.Val1147Ala 22160394:150:72
status: NEW
view ABCC7 p.Val1147Ala details
ABCC7 p.Asp1152Ala
X
ABCC7 p.Asp1152Ala 22160394:150:116
status: NEW
view ABCC7 p.Asp1152Ala details
ABCC7 p.Ile1151Ala
X
ABCC7 p.Ile1151Ala 22160394:150:104
status: NEW
view ABCC7 p.Ile1151Ala details
ABCC7 p.Asn1148Ala
X
ABCC7 p.Asn1148Ala 22160394:150:80
status: NEW
view ABCC7 p.Asn1148Ala details
ABCC7 p.Ser1150Ala
X
ABCC7 p.Ser1150Ala 22160394:150:96
status: NEW
view ABCC7 p.Ser1150Ala details
ABCC7 p.Met1140Ala
X
ABCC7 p.Met1140Ala 22160394:150:56
status: NEW
view ABCC7 p.Met1140Ala details
Surprisingly, nine mutations of TM12, including N1138A, M1140A, T1142A, V1147A, N1148A, S1149A, S1150A, I1151A, and D1152A, exhibited significantly altered block by Glyb; the pattern was not consistent with either α-helix or β-strand secondary structure along the full length of the region studied. Login to comment
151 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:151:136
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:151:337
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:151:557
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 22160394:151:154
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 22160394:151:355
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 22160394:151:575
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:151:160
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:151:361
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:151:581
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Thr339Ala
X
ABCC7 p.Thr339Ala 22160394:151:148
status: NEW
view ABCC7 p.Thr339Ala details
ABCC7 p.Thr339Ala
X
ABCC7 p.Thr339Ala 22160394:151:349
status: NEW
view ABCC7 p.Thr339Ala details
ABCC7 p.Thr339Ala
X
ABCC7 p.Thr339Ala 22160394:151:569
status: NEW
view ABCC7 p.Thr339Ala details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:151:172
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:151:373
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:151:593
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:151:178
status: NEW
view ABCC7 p.Arg334Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:151:379
status: NEW
view ABCC7 p.Arg334Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:151:599
status: NEW
view ABCC7 p.Arg334Ala details
ABCC7 p.Ile344Ala
X
ABCC7 p.Ile344Ala 22160394:151:118
status: NEW
view ABCC7 p.Ile344Ala details
ABCC7 p.Ile344Ala
X
ABCC7 p.Ile344Ala 22160394:151:319
status: NEW
view ABCC7 p.Ile344Ala details
ABCC7 p.Ile344Ala
X
ABCC7 p.Ile344Ala 22160394:151:539
status: NEW
view ABCC7 p.Ile344Ala details
ABCC7 p.Thr351Ala
X
ABCC7 p.Thr351Ala 22160394:151:76
status: NEW
view ABCC7 p.Thr351Ala details
ABCC7 p.Thr351Ala
X
ABCC7 p.Thr351Ala 22160394:151:277
status: NEW
view ABCC7 p.Thr351Ala details
ABCC7 p.Thr351Ala
X
ABCC7 p.Thr351Ala 22160394:151:497
status: NEW
view ABCC7 p.Thr351Ala details
ABCC7 p.Ile340Ala
X
ABCC7 p.Ile340Ala 22160394:151:142
status: NEW
view ABCC7 p.Ile340Ala details
ABCC7 p.Ile340Ala
X
ABCC7 p.Ile340Ala 22160394:151:343
status: NEW
view ABCC7 p.Ile340Ala details
ABCC7 p.Ile340Ala
X
ABCC7 p.Ile340Ala 22160394:151:563
status: NEW
view ABCC7 p.Ile340Ala details
ABCC7 p.Ile336Ala
X
ABCC7 p.Ile336Ala 22160394:151:166
status: NEW
view ABCC7 p.Ile336Ala details
ABCC7 p.Ile336Ala
X
ABCC7 p.Ile336Ala 22160394:151:367
status: NEW
view ABCC7 p.Ile336Ala details
ABCC7 p.Ile336Ala
X
ABCC7 p.Ile336Ala 22160394:151:587
status: NEW
view ABCC7 p.Ile336Ala details
ABCC7 p.Arg347Ala
X
ABCC7 p.Arg347Ala 22160394:151:100
status: NEW
view ABCC7 p.Arg347Ala details
ABCC7 p.Arg347Ala
X
ABCC7 p.Arg347Ala 22160394:151:301
status: NEW
view ABCC7 p.Arg347Ala details
ABCC7 p.Arg347Ala
X
ABCC7 p.Arg347Ala 22160394:151:521
status: NEW
view ABCC7 p.Arg347Ala details
ABCC7 p.Arg352Ala
X
ABCC7 p.Arg352Ala 22160394:151:70
status: NEW
view ABCC7 p.Arg352Ala details
ABCC7 p.Arg352Ala
X
ABCC7 p.Arg352Ala 22160394:151:271
status: NEW
view ABCC7 p.Arg352Ala details
ABCC7 p.Arg352Ala
X
ABCC7 p.Arg352Ala 22160394:151:491
status: NEW
view ABCC7 p.Arg352Ala details
ABCC7 p.Ala349Ser
X
ABCC7 p.Ala349Ser 22160394:151:88
status: NEW
view ABCC7 p.Ala349Ser details
ABCC7 p.Ala349Ser
X
ABCC7 p.Ala349Ser 22160394:151:289
status: NEW
view ABCC7 p.Ala349Ser details
ABCC7 p.Ala349Ser
X
ABCC7 p.Ala349Ser 22160394:151:509
status: NEW
view ABCC7 p.Ala349Ser details
ABCC7 p.Cys343Ala
X
ABCC7 p.Cys343Ala 22160394:151:124
status: NEW
view ABCC7 p.Cys343Ala details
ABCC7 p.Cys343Ala
X
ABCC7 p.Cys343Ala 22160394:151:325
status: NEW
view ABCC7 p.Cys343Ala details
ABCC7 p.Cys343Ala
X
ABCC7 p.Cys343Ala 22160394:151:545
status: NEW
view ABCC7 p.Cys343Ala details
ABCC7 p.Phe342Ala
X
ABCC7 p.Phe342Ala 22160394:151:130
status: NEW
view ABCC7 p.Phe342Ala details
ABCC7 p.Phe342Ala
X
ABCC7 p.Phe342Ala 22160394:151:331
status: NEW
view ABCC7 p.Phe342Ala details
ABCC7 p.Phe342Ala
X
ABCC7 p.Phe342Ala 22160394:151:551
status: NEW
view ABCC7 p.Phe342Ala details
ABCC7 p.Met348Ala
X
ABCC7 p.Met348Ala 22160394:151:94
status: NEW
view ABCC7 p.Met348Ala details
ABCC7 p.Met348Ala
X
ABCC7 p.Met348Ala 22160394:151:295
status: NEW
view ABCC7 p.Met348Ala details
ABCC7 p.Met348Ala
X
ABCC7 p.Met348Ala 22160394:151:515
status: NEW
view ABCC7 p.Met348Ala details
ABCC7 p.Val350Ala
X
ABCC7 p.Val350Ala 22160394:151:82
status: NEW
view ABCC7 p.Val350Ala details
ABCC7 p.Val350Ala
X
ABCC7 p.Val350Ala 22160394:151:283
status: NEW
view ABCC7 p.Val350Ala details
ABCC7 p.Val350Ala
X
ABCC7 p.Val350Ala 22160394:151:503
status: NEW
view ABCC7 p.Val350Ala details
ABCC7 p.Val345Ala
X
ABCC7 p.Val345Ala 22160394:151:112
status: NEW
view ABCC7 p.Val345Ala details
ABCC7 p.Val345Ala
X
ABCC7 p.Val345Ala 22160394:151:313
status: NEW
view ABCC7 p.Val345Ala details
ABCC7 p.Val345Ala
X
ABCC7 p.Val345Ala 22160394:151:533
status: NEW
view ABCC7 p.Val345Ala details
ABCC7 p.Gln353Ala
X
ABCC7 p.Gln353Ala 22160394:151:64
status: NEW
view ABCC7 p.Gln353Ala details
ABCC7 p.Gln353Ala
X
ABCC7 p.Gln353Ala 22160394:151:265
status: NEW
view ABCC7 p.Gln353Ala details
ABCC7 p.Gln353Ala
X
ABCC7 p.Gln353Ala 22160394:151:485
status: NEW
view ABCC7 p.Gln353Ala details
ABCC7 p.Leu346Ala
X
ABCC7 p.Leu346Ala 22160394:151:106
status: NEW
view ABCC7 p.Leu346Ala details
ABCC7 p.Leu346Ala
X
ABCC7 p.Leu346Ala 22160394:151:307
status: NEW
view ABCC7 p.Leu346Ala details
ABCC7 p.Leu346Ala
X
ABCC7 p.Leu346Ala 22160394:151:527
status: NEW
view ABCC7 p.Leu346Ala details
The surprising finding that mutations at six adjacent positions Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** * * * 0.8 0.6 0.4 0.2 0 Fractional block by Glyb50 μM Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** ** ** * * * * * * ** ** Fractional block by Tolb300 μM 0.8 0.6 0.4 0.2 0 Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT * ** ** ** ** ** ** ** ** Fractional block by Glip200 μM 0.8 0.6 0.4 0.2 0 Fig. 3 Alanine-scanning in TM6 to identify the amino acids that interact with the three blockers. Login to comment
157 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:157:66
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:157:76
status: NEW
view ABCC7 p.Phe337Ala details
Out of 20 mutants in TM6 and 20 mutants in TM12, only two in TM6 (S341A and F337A) induced rectification in macropatch currents which were suggested to form the narrow part of the pore (see below, Fig. 7, Supplementary Fig. 3). Login to comment
158 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:158:219
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:158:212
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:158:205
status: NEW
view ABCC7 p.Arg334Ala details
ABCC7 p.Arg347Ala
X
ABCC7 p.Arg347Ala 22160394:158:226
status: NEW
view ABCC7 p.Arg347Ala details
ABCC7 p.Arg352Ala
X
ABCC7 p.Arg352Ala 22160394:158:237
status: NEW
view ABCC7 p.Arg352Ala details
Among the 20 single amino acid mutants of TM12 that we tested in this paper, none of them exhibited significant change in their single-channel conductance compared to WT-CFTR, while we know that mutations R334A, F337A, S341A, R347A, and R352A in TM6 all exhibited significant change in their single-channel conductance [11, 12, 29, and the present manuscript]; these data strongly suggest that TM6 and TM12 do not equally contribute to the pore of CFTR. Login to comment
162 ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:162:29
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:162:19
status: NEW
view ABCC7 p.Arg334Ala details
Although mutations R334A and K335A exhibited opposite effects on steady-state block by Glyb and Glip, neither mutation altered initial block (Fig. 5). Login to comment
163 ABCC7 p.Asn1138Ala
X
ABCC7 p.Asn1138Ala 22160394:163:203
status: NEW
view ABCC7 p.Asn1138Ala details
ABCC7 p.Asn1138Ala
X
ABCC7 p.Asn1138Ala 22160394:163:386
status: NEW
view ABCC7 p.Asn1138Ala details
ABCC7 p.Thr1134Ala
X
ABCC7 p.Thr1134Ala 22160394:163:231
status: NEW
view ABCC7 p.Thr1134Ala details
ABCC7 p.Thr1134Ala
X
ABCC7 p.Thr1134Ala 22160394:163:414
status: NEW
view ABCC7 p.Thr1134Ala details
ABCC7 p.Ser1141Ala
X
ABCC7 p.Ser1141Ala 22160394:163:182
status: NEW
view ABCC7 p.Ser1141Ala details
ABCC7 p.Ser1141Ala
X
ABCC7 p.Ser1141Ala 22160394:163:365
status: NEW
view ABCC7 p.Ser1141Ala details
ABCC7 p.Met1137Ala
X
ABCC7 p.Met1137Ala 22160394:163:210
status: NEW
view ABCC7 p.Met1137Ala details
ABCC7 p.Met1137Ala
X
ABCC7 p.Met1137Ala 22160394:163:393
status: NEW
view ABCC7 p.Met1137Ala details
ABCC7 p.Thr1142Ala
X
ABCC7 p.Thr1142Ala 22160394:163:175
status: NEW
view ABCC7 p.Thr1142Ala details
ABCC7 p.Thr1142Ala
X
ABCC7 p.Thr1142Ala 22160394:163:358
status: NEW
view ABCC7 p.Thr1142Ala details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:163:55
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:163:45
status: NEW
view ABCC7 p.Arg334Ala details
ABCC7 p.Ser1149Ala
X
ABCC7 p.Ser1149Ala 22160394:163:126
status: NEW
view ABCC7 p.Ser1149Ala details
ABCC7 p.Ser1149Ala
X
ABCC7 p.Ser1149Ala 22160394:163:309
status: NEW
view ABCC7 p.Ser1149Ala details
ABCC7 p.Val1147Ala
X
ABCC7 p.Val1147Ala 22160394:163:140
status: NEW
view ABCC7 p.Val1147Ala details
ABCC7 p.Val1147Ala
X
ABCC7 p.Val1147Ala 22160394:163:323
status: NEW
view ABCC7 p.Val1147Ala details
ABCC7 p.Asp1152Ala
X
ABCC7 p.Asp1152Ala 22160394:163:105
status: NEW
view ABCC7 p.Asp1152Ala details
ABCC7 p.Asp1152Ala
X
ABCC7 p.Asp1152Ala 22160394:163:288
status: NEW
view ABCC7 p.Asp1152Ala details
ABCC7 p.Ile1151Ala
X
ABCC7 p.Ile1151Ala 22160394:163:112
status: NEW
view ABCC7 p.Ile1151Ala details
ABCC7 p.Ile1151Ala
X
ABCC7 p.Ile1151Ala 22160394:163:295
status: NEW
view ABCC7 p.Ile1151Ala details
ABCC7 p.Asn1148Ala
X
ABCC7 p.Asn1148Ala 22160394:163:133
status: NEW
view ABCC7 p.Asn1148Ala details
ABCC7 p.Asn1148Ala
X
ABCC7 p.Asn1148Ala 22160394:163:316
status: NEW
view ABCC7 p.Asn1148Ala details
ABCC7 p.Ser1150Ala
X
ABCC7 p.Ser1150Ala 22160394:163:119
status: NEW
view ABCC7 p.Ser1150Ala details
ABCC7 p.Ser1150Ala
X
ABCC7 p.Ser1150Ala 22160394:163:302
status: NEW
view ABCC7 p.Ser1150Ala details
ABCC7 p.Met1140Ala
X
ABCC7 p.Met1140Ala 22160394:163:189
status: NEW
view ABCC7 p.Met1140Ala details
ABCC7 p.Met1140Ala
X
ABCC7 p.Met1140Ala 22160394:163:372
status: NEW
view ABCC7 p.Met1140Ala details
ABCC7 p.Ile1139Ala
X
ABCC7 p.Ile1139Ala 22160394:163:196
status: NEW
view ABCC7 p.Ile1139Ala details
ABCC7 p.Ile1139Ala
X
ABCC7 p.Ile1139Ala 22160394:163:379
status: NEW
view ABCC7 p.Ile1139Ala details
ABCC7 p.Leu1143Ala
X
ABCC7 p.Leu1143Ala 22160394:163:168
status: NEW
view ABCC7 p.Leu1143Ala details
ABCC7 p.Leu1143Ala
X
ABCC7 p.Leu1143Ala 22160394:163:351
status: NEW
view ABCC7 p.Leu1143Ala details
ABCC7 p.Leu1135Ala
X
ABCC7 p.Leu1135Ala 22160394:163:224
status: NEW
view ABCC7 p.Leu1135Ala details
ABCC7 p.Leu1135Ala
X
ABCC7 p.Leu1135Ala 22160394:163:407
status: NEW
view ABCC7 p.Leu1135Ala details
ABCC7 p.Trp1145Ala
X
ABCC7 p.Trp1145Ala 22160394:163:154
status: NEW
view ABCC7 p.Trp1145Ala details
ABCC7 p.Trp1145Ala
X
ABCC7 p.Trp1145Ala 22160394:163:337
status: NEW
view ABCC7 p.Trp1145Ala details
ABCC7 p.Ala1136Ser
X
ABCC7 p.Ala1136Ser 22160394:163:217
status: NEW
view ABCC7 p.Ala1136Ser details
ABCC7 p.Ala1136Ser
X
ABCC7 p.Ala1136Ser 22160394:163:400
status: NEW
view ABCC7 p.Ala1136Ser details
ABCC7 p.Gln1144Ala
X
ABCC7 p.Gln1144Ala 22160394:163:161
status: NEW
view ABCC7 p.Gln1144Ala details
ABCC7 p.Gln1144Ala
X
ABCC7 p.Gln1144Ala 22160394:163:344
status: NEW
view ABCC7 p.Gln1144Ala details
ABCC7 p.Ala1146Ser
X
ABCC7 p.Ala1146Ser 22160394:163:147
status: NEW
view ABCC7 p.Ala1146Ser details
ABCC7 p.Ala1146Ser
X
ABCC7 p.Ala1146Ser 22160394:163:330
status: NEW
view ABCC7 p.Ala1146Ser details
ABCC7 p.Val1153Ala
X
ABCC7 p.Val1153Ala 22160394:163:98
status: NEW
view ABCC7 p.Val1153Ala details
ABCC7 p.Val1153Ala
X
ABCC7 p.Val1153Ala 22160394:163:281
status: NEW
view ABCC7 p.Val1153Ala details
Effects on time-dependent block by mutations R334A and K335A Fractional block by Glip200 μM V1153A D1152A I1151A S1150A S1149A N1148A V1147A A1146S W1145A Q1144A L1143A T1142A S1141A M1140A I1139A N1138A M1137A A1136S L1135A T1134A WT 0 0.2 0.4 0.6 0.8 * ** ** ** ** ** ** * V1153A D1152A I1151A S1150A S1149A N1148A V1147A A1146S W1145A Q1144A L1143A T1142A S1141A M1140A I1139A N1138A M1137A A1136S L1135A T1134A WT 0 0.2 0.4 0.6 0.8 1.0 * * * * * ** ** ** ** Fractional block by Glyb50 μM Fig. 4 Alanine-scanning in TM12 to identify amino acids that interact with Glyb and Glip. Login to comment
166 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:166:151
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:166:336
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 22160394:166:169
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 22160394:166:354
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:166:175
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:166:360
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Thr339Ala
X
ABCC7 p.Thr339Ala 22160394:166:163
status: NEW
view ABCC7 p.Thr339Ala details
ABCC7 p.Thr339Ala
X
ABCC7 p.Thr339Ala 22160394:166:348
status: NEW
view ABCC7 p.Thr339Ala details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:166:187
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:166:372
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:166:193
status: NEW
view ABCC7 p.Arg334Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:166:378
status: NEW
view ABCC7 p.Arg334Ala details
ABCC7 p.Ile344Ala
X
ABCC7 p.Ile344Ala 22160394:166:133
status: NEW
view ABCC7 p.Ile344Ala details
ABCC7 p.Ile344Ala
X
ABCC7 p.Ile344Ala 22160394:166:318
status: NEW
view ABCC7 p.Ile344Ala details
ABCC7 p.Thr351Ala
X
ABCC7 p.Thr351Ala 22160394:166:91
status: NEW
view ABCC7 p.Thr351Ala details
ABCC7 p.Thr351Ala
X
ABCC7 p.Thr351Ala 22160394:166:276
status: NEW
view ABCC7 p.Thr351Ala details
ABCC7 p.Ile340Ala
X
ABCC7 p.Ile340Ala 22160394:166:157
status: NEW
view ABCC7 p.Ile340Ala details
ABCC7 p.Ile340Ala
X
ABCC7 p.Ile340Ala 22160394:166:342
status: NEW
view ABCC7 p.Ile340Ala details
ABCC7 p.Ile336Ala
X
ABCC7 p.Ile336Ala 22160394:166:181
status: NEW
view ABCC7 p.Ile336Ala details
ABCC7 p.Ile336Ala
X
ABCC7 p.Ile336Ala 22160394:166:366
status: NEW
view ABCC7 p.Ile336Ala details
ABCC7 p.Arg347Ala
X
ABCC7 p.Arg347Ala 22160394:166:115
status: NEW
view ABCC7 p.Arg347Ala details
ABCC7 p.Arg347Ala
X
ABCC7 p.Arg347Ala 22160394:166:300
status: NEW
view ABCC7 p.Arg347Ala details
ABCC7 p.Arg352Ala
X
ABCC7 p.Arg352Ala 22160394:166:85
status: NEW
view ABCC7 p.Arg352Ala details
ABCC7 p.Arg352Ala
X
ABCC7 p.Arg352Ala 22160394:166:270
status: NEW
view ABCC7 p.Arg352Ala details
ABCC7 p.Ala349Ser
X
ABCC7 p.Ala349Ser 22160394:166:103
status: NEW
view ABCC7 p.Ala349Ser details
ABCC7 p.Ala349Ser
X
ABCC7 p.Ala349Ser 22160394:166:288
status: NEW
view ABCC7 p.Ala349Ser details
ABCC7 p.Cys343Ala
X
ABCC7 p.Cys343Ala 22160394:166:139
status: NEW
view ABCC7 p.Cys343Ala details
ABCC7 p.Cys343Ala
X
ABCC7 p.Cys343Ala 22160394:166:324
status: NEW
view ABCC7 p.Cys343Ala details
ABCC7 p.Phe342Ala
X
ABCC7 p.Phe342Ala 22160394:166:145
status: NEW
view ABCC7 p.Phe342Ala details
ABCC7 p.Phe342Ala
X
ABCC7 p.Phe342Ala 22160394:166:330
status: NEW
view ABCC7 p.Phe342Ala details
ABCC7 p.Met348Ala
X
ABCC7 p.Met348Ala 22160394:166:109
status: NEW
view ABCC7 p.Met348Ala details
ABCC7 p.Met348Ala
X
ABCC7 p.Met348Ala 22160394:166:294
status: NEW
view ABCC7 p.Met348Ala details
ABCC7 p.Val350Ala
X
ABCC7 p.Val350Ala 22160394:166:97
status: NEW
view ABCC7 p.Val350Ala details
ABCC7 p.Val350Ala
X
ABCC7 p.Val350Ala 22160394:166:282
status: NEW
view ABCC7 p.Val350Ala details
ABCC7 p.Val345Ala
X
ABCC7 p.Val345Ala 22160394:166:127
status: NEW
view ABCC7 p.Val345Ala details
ABCC7 p.Val345Ala
X
ABCC7 p.Val345Ala 22160394:166:312
status: NEW
view ABCC7 p.Val345Ala details
ABCC7 p.Gln353Ala
X
ABCC7 p.Gln353Ala 22160394:166:79
status: NEW
view ABCC7 p.Gln353Ala details
ABCC7 p.Gln353Ala
X
ABCC7 p.Gln353Ala 22160394:166:264
status: NEW
view ABCC7 p.Gln353Ala details
ABCC7 p.Leu346Ala
X
ABCC7 p.Leu346Ala 22160394:166:121
status: NEW
view ABCC7 p.Leu346Ala details
ABCC7 p.Leu346Ala
X
ABCC7 p.Leu346Ala 22160394:166:306
status: NEW
view ABCC7 p.Leu346Ala details
Double asterisks indicate significantly different compared to WT-CFTR (p<0.01) Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.3 0.2 0.1 0 * * ** ** 0.4 Initial block by 50 μM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.4 0.3 0.2 0.1 0 ** ** * Initial block by 200 μM Glip Fig. 5 Initial block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in steady-state fractional block according to Fig. 3 (bars show mean±SEM, n=5-10). Login to comment
168 ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:168:192
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:168:135
status: NEW
view ABCC7 p.Arg334Ala details
Double asterisks indicate significantly different compared to WT-CFTR (p<0.01) were similar for Glyb and Glip, although the effect of R334A on Glyb was larger than for Glip and the effect of K335A was larger for Glip than Glyb (Fig. 6). Login to comment
170 ABCC7 p.Met348Ala
X
ABCC7 p.Met348Ala 22160394:170:10
status: NEW
view ABCC7 p.Met348Ala details
ABCC7 p.Val350Ala
X
ABCC7 p.Val350Ala 22160394:170:20
status: NEW
view ABCC7 p.Val350Ala details
Mutations M348A and V350A at sites predicted to lie in the inner vestibule strongly increased steady-state block of CFTR by Glyb and Glip (Fig. 3). Login to comment
171 ABCC7 p.Met348Ala
X
ABCC7 p.Met348Ala 22160394:171:3
status: NEW
view ABCC7 p.Met348Ala details
ABCC7 p.Val350Ala
X
ABCC7 p.Val350Ala 22160394:171:117
status: NEW
view ABCC7 p.Val350Ala details
In M348A, the initial component of block was nearly lost for both Glyb and Glip; this effect was somewhat smaller in V350A (Fig. 5). Login to comment
173 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:173:9
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Arg347Ala
X
ABCC7 p.Arg347Ala 22160394:173:81
status: NEW
view ABCC7 p.Arg347Ala details
ABCC7 p.Arg352Ala
X
ABCC7 p.Arg352Ala 22160394:173:91
status: NEW
view ABCC7 p.Arg352Ala details
Mutation S341A caused the largest decrease in block by Glyb and Glip (aside from R347A and R352A, which have non-canonical effects as described above; Fig. 3). Login to comment
175 ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:175:9
status: NEW
view ABCC7 p.Phe337Ala details
Mutation F337A caused a significant decrease in block by Glyb but a significant increase in block by Glip (Fig. 3). Login to comment
176 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:176:39
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:176:80
status: NEW
view ABCC7 p.Phe337Ala details
In contrast to the effects of mutation S341A, the reduction in block by Glyb in F337A reflected a substantial decrease in initial block without a change in the magnitude of time-dependent block (Figs. 5 and 6). Login to comment
177 ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:177:36
status: NEW
view ABCC7 p.Phe337Ala details
Meanwhile, the increase in block of F337A by Glip reflected a clear decrease in initial block with a dramatic increase in the magnitude of time-dependent block (Figs. 5 and 6). Login to comment
178 ABCC7 p.Val350Ala
X
ABCC7 p.Val350Ala 22160394:178:114
status: NEW
view ABCC7 p.Val350Ala details
ABCC7 p.Val350Ala
X
ABCC7 p.Val350Ala 22160394:178:373
status: NEW
view ABCC7 p.Val350Ala details
The apparent voltage-dependence of block by Glyb and Glip was not altered in any of the TM6 mutants other than in V350A suggesting that although several of these mutations affected the magnitude and kinetics of inhibition, the position of the blocker molecule in the voltage field at steady-state was not significantly different from that in the wildtype channel except in V350A (Fig. 8a, b). Login to comment
179 ABCC7 p.Met348Ala
X
ABCC7 p.Met348Ala 22160394:179:5
status: NEW
view ABCC7 p.Met348Ala details
ABCC7 p.Val350Ala
X
ABCC7 p.Val350Ala 22160394:179:15
status: NEW
view ABCC7 p.Val350Ala details
Both M348A and V350A single-channel full conductances are similar to that of WT-CFTR (Fig. 9, data not shown). Login to comment
180 ABCC7 p.Ser1149Ala
X
ABCC7 p.Ser1149Ala 22160394:180:64
status: NEW
view ABCC7 p.Ser1149Ala details
ABCC7 p.Val1147Ala
X
ABCC7 p.Val1147Ala 22160394:180:48
status: NEW
view ABCC7 p.Val1147Ala details
ABCC7 p.Ile1151Ala
X
ABCC7 p.Ile1151Ala 22160394:180:84
status: NEW
view ABCC7 p.Ile1151Ala details
ABCC7 p.Asn1148Ala
X
ABCC7 p.Asn1148Ala 22160394:180:56
status: NEW
view ABCC7 p.Asn1148Ala details
ABCC7 p.Ser1150Ala
X
ABCC7 p.Ser1150Ala 22160394:180:72
status: NEW
view ABCC7 p.Ser1150Ala details
ABCC7 p.Gln1144Ala
X
ABCC7 p.Gln1144Ala 22160394:180:40
status: NEW
view ABCC7 p.Gln1144Ala details
Surprisingly, several mutations in TM12 Q1144A, V1147A, N1148A, S1149A, S1150A, and I1151A affected the voltage-dependence of block by Glyb (Fig. 8b). Login to comment
183 ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:183:180
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 22160394:183:0
status: NEW
view ABCC7 p.Phe337Cys details
ABCC7 p.Phe337Glu
X
ABCC7 p.Phe337Glu 22160394:183:10
status: NEW
view ABCC7 p.Phe337Glu details
F337C-and F337E-CFTR exhibited significantly altered reversal potential, relative permeability, and relative conductance compared to WT-CFTR (Supplementary Tables 1, 2, 3), as did F337A-, S-, Y-, and L-CFTR [24]. Login to comment
184 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:184:15
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:184:25
status: NEW
view ABCC7 p.Phe337Ala details
Both mutations S341A and F337A significantly decreased single-channel conductance (Fig. 9 and Ref. [29]). Login to comment
185 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:185:67
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:185:110
status: NEW
view ABCC7 p.Phe337Ala details
Consistent with this designation, macroscopic chloride currents in S341A exhibited inward rectification while F337A/C/E exhibited outward rectification (Fig. 7; Supplementary Fig. 1) [28, 29]. Login to comment
190 ABCC7 p.Asp1152Ala
X
ABCC7 p.Asp1152Ala 22160394:190:28
status: NEW
view ABCC7 p.Asp1152Ala details
All of these mutants except D1152A showed stable open-closed behavior similar to that of human WT-CFTR with subconductance states as rare events. Login to comment
191 ABCC7 p.Asp1152Ala
X
ABCC7 p.Asp1152Ala 22160394:191:0
status: NEW
view ABCC7 p.Asp1152Ala details
D1152A exhibited increased single-channel conductance for the full open state while it also showed frequent transitions to subconductance states (-0.98±0.02 pA, n=4; Fig. 9). Login to comment
192 ABCC7 p.Thr1134Ala
X
ABCC7 p.Thr1134Ala 22160394:192:47
status: NEW
view ABCC7 p.Thr1134Ala details
ABCC7 p.Ser1141Ala
X
ABCC7 p.Ser1141Ala 22160394:192:0
status: NEW
view ABCC7 p.Ser1141Ala details
S1141A (-0.83±0.02 pA, n=5) increased and T1134A (-0.59±0.02 pA, n=4) decreased single-channel full open state amplitude compared to WT-CFTR (-0.70±0.03 pA, n=10; Fig. 9). Login to comment
193 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:193:235
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:193:432
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 22160394:193:253
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 22160394:193:450
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:193:259
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:193:456
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Thr339Ala
X
ABCC7 p.Thr339Ala 22160394:193:247
status: NEW
view ABCC7 p.Thr339Ala details
ABCC7 p.Thr339Ala
X
ABCC7 p.Thr339Ala 22160394:193:444
status: NEW
view ABCC7 p.Thr339Ala details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:193:271
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:193:468
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:193:277
status: NEW
view ABCC7 p.Arg334Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:193:474
status: NEW
view ABCC7 p.Arg334Ala details
ABCC7 p.Ile344Ala
X
ABCC7 p.Ile344Ala 22160394:193:217
status: NEW
view ABCC7 p.Ile344Ala details
ABCC7 p.Ile344Ala
X
ABCC7 p.Ile344Ala 22160394:193:414
status: NEW
view ABCC7 p.Ile344Ala details
ABCC7 p.Thr351Ala
X
ABCC7 p.Thr351Ala 22160394:193:175
status: NEW
view ABCC7 p.Thr351Ala details
ABCC7 p.Thr351Ala
X
ABCC7 p.Thr351Ala 22160394:193:372
status: NEW
view ABCC7 p.Thr351Ala details
ABCC7 p.Ile340Ala
X
ABCC7 p.Ile340Ala 22160394:193:241
status: NEW
view ABCC7 p.Ile340Ala details
ABCC7 p.Ile340Ala
X
ABCC7 p.Ile340Ala 22160394:193:438
status: NEW
view ABCC7 p.Ile340Ala details
ABCC7 p.Ile336Ala
X
ABCC7 p.Ile336Ala 22160394:193:265
status: NEW
view ABCC7 p.Ile336Ala details
ABCC7 p.Ile336Ala
X
ABCC7 p.Ile336Ala 22160394:193:462
status: NEW
view ABCC7 p.Ile336Ala details
ABCC7 p.Arg347Ala
X
ABCC7 p.Arg347Ala 22160394:193:199
status: NEW
view ABCC7 p.Arg347Ala details
ABCC7 p.Arg347Ala
X
ABCC7 p.Arg347Ala 22160394:193:396
status: NEW
view ABCC7 p.Arg347Ala details
ABCC7 p.Arg352Ala
X
ABCC7 p.Arg352Ala 22160394:193:169
status: NEW
view ABCC7 p.Arg352Ala details
ABCC7 p.Arg352Ala
X
ABCC7 p.Arg352Ala 22160394:193:366
status: NEW
view ABCC7 p.Arg352Ala details
ABCC7 p.Ala349Ser
X
ABCC7 p.Ala349Ser 22160394:193:187
status: NEW
view ABCC7 p.Ala349Ser details
ABCC7 p.Ala349Ser
X
ABCC7 p.Ala349Ser 22160394:193:384
status: NEW
view ABCC7 p.Ala349Ser details
ABCC7 p.Cys343Ala
X
ABCC7 p.Cys343Ala 22160394:193:223
status: NEW
view ABCC7 p.Cys343Ala details
ABCC7 p.Cys343Ala
X
ABCC7 p.Cys343Ala 22160394:193:420
status: NEW
view ABCC7 p.Cys343Ala details
ABCC7 p.Phe342Ala
X
ABCC7 p.Phe342Ala 22160394:193:229
status: NEW
view ABCC7 p.Phe342Ala details
ABCC7 p.Phe342Ala
X
ABCC7 p.Phe342Ala 22160394:193:426
status: NEW
view ABCC7 p.Phe342Ala details
ABCC7 p.Met348Ala
X
ABCC7 p.Met348Ala 22160394:193:193
status: NEW
view ABCC7 p.Met348Ala details
ABCC7 p.Met348Ala
X
ABCC7 p.Met348Ala 22160394:193:390
status: NEW
view ABCC7 p.Met348Ala details
ABCC7 p.Val350Ala
X
ABCC7 p.Val350Ala 22160394:193:181
status: NEW
view ABCC7 p.Val350Ala details
ABCC7 p.Val350Ala
X
ABCC7 p.Val350Ala 22160394:193:378
status: NEW
view ABCC7 p.Val350Ala details
ABCC7 p.Val345Ala
X
ABCC7 p.Val345Ala 22160394:193:211
status: NEW
view ABCC7 p.Val345Ala details
ABCC7 p.Val345Ala
X
ABCC7 p.Val345Ala 22160394:193:408
status: NEW
view ABCC7 p.Val345Ala details
ABCC7 p.Gln353Ala
X
ABCC7 p.Gln353Ala 22160394:193:163
status: NEW
view ABCC7 p.Gln353Ala details
ABCC7 p.Gln353Ala
X
ABCC7 p.Gln353Ala 22160394:193:360
status: NEW
view ABCC7 p.Gln353Ala details
ABCC7 p.Leu346Ala
X
ABCC7 p.Leu346Ala 22160394:193:205
status: NEW
view ABCC7 p.Leu346Ala details
ABCC7 p.Leu346Ala
X
ABCC7 p.Leu346Ala 22160394:193:402
status: NEW
view ABCC7 p.Leu346Ala details
Probable orientation of drugs in the pore Glyb and Glip are identical molecules along most of their lengths, differing only in the substituents on the ring at the Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.8 0.6 0.2 0 ** ** ** ** Time-dependent block by 50 μμM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** * ** * Time-dependent block by 200 μM Glip 0.4 0.8 0.6 0.2 00.4 Fig. 6 Time-dependent block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in fractional block according to Fig. 3 (bars show mean±SEM, n=5-10). Login to comment
196 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:196:49
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:196:59
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Met348Ala
X
ABCC7 p.Met348Ala 22160394:196:135
status: NEW
view ABCC7 p.Met348Ala details
ABCC7 p.Val350Ala
X
ABCC7 p.Val350Ala 22160394:196:145
status: NEW
view ABCC7 p.Val350Ala details
From the differences in the effects of mutations S341A and F337A on block by Glyb and Glip, and the similarity of effects of mutations M348A and V350A on block by the two drugs, we can infer that both drugs bind in the pore with the sulfonylurea-linked cyclohexamide end facing toward the cytoplasm. Login to comment
201 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:201:52
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:201:62
status: NEW
view ABCC7 p.Phe337Ala details
Similar to their effects on block by Glyb, both the S341A and F337A mutations decreased the efficacy of block by Meglitinide (fractional block was 0.35±0.04 and 0.45±0.04, p<0.01, respectively). Login to comment
206 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:206:106
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:206:112
status: NEW
view ABCC7 p.Phe337Ala details
Each of the functional parameters comprising the biophysical signature of CFTR (single-channel conduc- WT S341A F337A Vm(mV) -100 -50 50 100 -1500 -1000 -500 500 1000 1500 I (pA) ATP -100 -50 50 100 -2000 -1000 1000 2000 ATP ATP +Glyb 50 μM ATP +Glyb 50 μMATP +Glyb 50 μM I (pA) Vm(mV) Vm(mV) -100 -50 50 100 -2000 -1000 1000 2000 ATP I (pA) Vm (mV) -100 -50 50 100 -2000 -1000 1000 2000 50 100 I (pA) ATP Vm(mV) -100 -50 50 100 -1500 -1000 -500 500 1000 1500 I (pA) ATP Vm(mV) -100 -50 50 100 -1500 -1000 -500 500 1000 1500 I (pA) ATP Vm(mV) -100 -50 50 100 -800 -400 400 800 I (pA) ATP Vm(mV) -100 -50 50 100 -800 -400 400 800 I (pA) ATP ATP +Glip 200 μMATP +Glip 200 μMATP +Glip 200 μM I (pA) Vm(mV) -100 -50 50 100 -800 -400 400 800 ATP ATP +Tolb 300 μM ATP +Tolb 300 μMATP +Tolb 300 μM Fig. 7 I-V relationships for WT-CFTR and two important mutants, from inside-out macropatches in symmetrical 150 mM Cl- solution. Data were obtained by ramping the membrane potential from VM=-100 mV to +100 mV over 300 ms. Login to comment
208 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:208:66
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:208:77
status: NEW
view ABCC7 p.Phe337Ala details
Data for each CFTR variant is from a single patch expressing WT-, S341A-, or F337A-CFTR tance, rectification, selectivity, blocker pharmacology, etc.) can be compared between wildtype and site-directed mutants to infer channel structure. Login to comment
219 ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 22160394:219:42
status: NEW
view ABCC7 p.Thr338Ala details
Gupta and Linsdell reported that mutation T338A reduced block by Glyb [21], but this was not the case in our experiments with either Glyb or Glip. Login to comment
220 ABCC7 p.Thr351Ala
X
ABCC7 p.Thr351Ala 22160394:220:58
status: NEW
view ABCC7 p.Thr351Ala details
ABCC7 p.Gln353Ala
X
ABCC7 p.Gln353Ala 22160394:220:67
status: NEW
view ABCC7 p.Gln353Ala details
Moreover, block by neither drug was affected by mutations T351A or Q353A, suggesting that these sites do not contribute to the region comprising the anion selectivity filter as previously suggested [9, 20]. Login to comment
221 ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:221:35
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 22160394:221:25
status: NEW
view ABCC7 p.Arg334Ala details
The effects of mutations R334A and K335A are indirect, likely related to the movement of chloride within the pore, or the stabilization of the outer vestibule. Login to comment
222 ABCC7 p.Arg347Ala
X
ABCC7 p.Arg347Ala 22160394:222:35
status: NEW
view ABCC7 p.Arg347Ala details
ABCC7 p.Arg352Ala
X
ABCC7 p.Arg352Ala 22160394:222:45
status: NEW
view ABCC7 p.Arg352Ala details
Likewise, the effects of mutations R347A and R352A are also indirect, because charge-destroying substitutions at these sites alter the gross architecture of the pore, with pleiotropic effects [11, 12]. Login to comment
224 ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:224:122
status: NEW
view ABCC7 p.Phe337Ala details
At V350, M348, and S341, alanine substitutions affected block by Glyb and Glip in an identical manner; the effects of the F337A mutation were opposite for Glyb and Glip. Login to comment
225 ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:225:355
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Thr1142Ala
X
ABCC7 p.Thr1142Ala 22160394:225:381
status: NEW
view ABCC7 p.Thr1142Ala details
Since these two drugs differ only at the non-sulfonylurea end of the molecular structure, it seems reasonable to conclude that it is this end of Glyb ΔFractionalblockfrom -20mVto-100mV ΔFractionalblockfrom -20mVto-100mV 0.0 0.1 0.2 0.3 0.4 0.5 * * #Glyb 0.0 0.1 0.2 0.3 0.4 0.5 * * * * * ## Glyb Vm(mV) -100 0.2 0.4 0.6 0.8 WT-Glyb50 μM F337A WT-Glyb100 μM T1142A Fractionalblock by50μMGlyb b a -200-40-60-80 Fig. 8 Voltage-dependent block of WT-CFTR and some important mutants in TM6 and TM12. Login to comment
226 ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:226:42
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Thr1142Ala
X
ABCC7 p.Thr1142Ala 22160394:226:58
status: NEW
view ABCC7 p.Thr1142Ala details
a Voltage-dependence of block of WT-CFTR, F337A-CFTR, and T1142A-CFTR by 50 μM Glyb, and WT-CFTR by 100 μM Glyb, at VM=-100 mV to -20 mV. Fractional block was calculated from the steady-state currents at each potential. Login to comment
231 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:231:74
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:231:84
status: NEW
view ABCC7 p.Phe337Ala details
This conclusion is bolstered by the finding that the effects of mutations S341A and F337A on block by Glyb were the same as their effects on block by Meglitinide, which shares structure with the non-sulfonylurea end of Glyb. Login to comment
232 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:232:187
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:232:167
status: NEW
view ABCC7 p.Phe337Ala details
In conclusion with these results, for the following reasons, we believe that the narrow region in TM6 of the CFTR pore is located between F337 and S341: (1) mutations F337A/S/C/E/Y/L and S341A/E/T dramatically altered the relative permeability of different anions in the channel (Supplementary Tables 2, 3; Refs. Login to comment
235 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:235:103
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:235:113
status: NEW
view ABCC7 p.Phe337Ala details
(3) Both F337 and S341 mutations exhibited outward or inward rectification, respectively; and (4) both S341A and F337A affected block by all four sulfonylurea family blockers [8, 21, 40, 42, 50, 53]. Login to comment
238 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 22160394:238:209
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:238:219
status: NEW
view ABCC7 p.Phe337Ala details
Therefore, we cannot conclude that one part of the Glyb molecule binds exclusively to one section of the pore because: (a) mutations along the full length of the pore affected block by Tolb, and (b) mutations S341A and F337A affected block by both Tolb and Meglitinide, which represent the two disparate halves of the Glyb structure. Login to comment
239 ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:239:198
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:239:790
status: NEW
view ABCC7 p.Phe337Ala details
ABCC7 p.Thr1134Ala
X
ABCC7 p.Thr1134Ala 22160394:239:265
status: NEW
view ABCC7 p.Thr1134Ala details
ABCC7 p.Thr1134Ala
X
ABCC7 p.Thr1134Ala 22160394:239:806
status: NEW
view ABCC7 p.Thr1134Ala details
ABCC7 p.Ser1141Ala
X
ABCC7 p.Ser1141Ala 22160394:239:287
status: NEW
view ABCC7 p.Ser1141Ala details
ABCC7 p.Ser1141Ala
X
ABCC7 p.Ser1141Ala 22160394:239:815
status: NEW
view ABCC7 p.Ser1141Ala details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:239:219
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 22160394:239:782
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Met348Ala
X
ABCC7 p.Met348Ala 22160394:239:177
status: NEW
view ABCC7 p.Met348Ala details
ABCC7 p.Met348Ala
X
ABCC7 p.Met348Ala 22160394:239:798
status: NEW
view ABCC7 p.Met348Ala details
ABCC7 p.Asp1152Ala
X
ABCC7 p.Asp1152Ala 22160394:239:247
status: NEW
view ABCC7 p.Asp1152Ala details
ABCC7 p.Asp1152Ala
X
ABCC7 p.Asp1152Ala 22160394:239:828
status: NEW
view ABCC7 p.Asp1152Ala details
Hence, strong time-dependent block of macropatch currents, and the appearance of multiple drug-induced closed states in single-channel recordings, may not arise from 0.4 pA 2 s M348A c f 0.2 pA 2 s F337A c f 0.4 pA 2 s K335A c f 0.4 pA 2 s c s2 f D1152A 0.4 pA 2 s T1134A c f 0.4 pA 2 s S1141A c f s2 0.4 pA 2 s c f WT 2000 4000 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 3000 9000 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 6000 400 1200 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 800 1600 1000 3000 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 2000 500 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 1000 4000 12000 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 8000 200 600 #ofevents 0.0 -0.5 -1.0 Current (pA) -1.50.5 400 Fig. 9 Representative single-channel traces for WT-, K335A-, F337A-, M348A-, T1134A-, S1141A-, and D1152A-CFTR (left) from excised inside-out membrane patches with symmetrical 150 mM Cl- solution, and their all-points amplitude histograms (right). Login to comment
243 ABCC7 p.Phe337Ala
X
ABCC7 p.Phe337Ala 22160394:243:71
status: NEW
view ABCC7 p.Phe337Ala details
The scale of the abscissa (current amplitude) in the current trace for F337A is different from the others interactions of a specific moiety with distinct binding sites in the pore [46-51]. Login to comment