ABCMdb

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 ABCC7 p.Arg334Ala For simplification, names of mutant channels are often abbreviated (e.g., R334A is R334A-CFTR). Login to comment 119 ABCC7 p.Ser341Ala ABCC7 p.Phe337Ala ABCC7 p.Lys335Ala ABCC7 p.Arg334Ala ABCC7 p.Ile344Ala ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala ABCC7 p.Met348Ala ABCC7 p.Val350Ala 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 ABCC7 p.Val345Ala 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 ABCC7 p.Arg334Ala 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 ABCC7 p.Arg334Ala R334A weakened block while K335A strengthened block by both blockers. Login to comment 135 ABCC7 p.Arg334Cys ABCC7 p.Arg334Ala 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 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 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 ABCC7 p.Arg352Ala Mutations R347A and R352A also represent a separate category from the rest. Login to comment 144 ABCC7 p.Asp993Arg ABCC7 p.Arg352Glu 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 ABCC7 p.Arg352Ala 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 ABCC7 p.Thr1142Ala ABCC7 p.Ser1149Ala ABCC7 p.Val1147Ala ABCC7 p.Asp1152Ala ABCC7 p.Ile1151Ala ABCC7 p.Asn1148Ala ABCC7 p.Ser1150Ala ABCC7 p.Met1140Ala 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 ABCC7 p.Ser341Ala ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala ABCC7 p.Thr339Ala ABCC7 p.Thr339Ala ABCC7 p.Thr339Ala ABCC7 p.Lys335Ala ABCC7 p.Lys335Ala ABCC7 p.Lys335Ala ABCC7 p.Arg334Ala ABCC7 p.Arg334Ala ABCC7 p.Arg334Ala ABCC7 p.Ile344Ala ABCC7 p.Ile344Ala ABCC7 p.Ile344Ala ABCC7 p.Thr351Ala ABCC7 p.Thr351Ala ABCC7 p.Thr351Ala ABCC7 p.Ile340Ala ABCC7 p.Ile340Ala ABCC7 p.Ile340Ala ABCC7 p.Ile336Ala ABCC7 p.Ile336Ala ABCC7 p.Ile336Ala ABCC7 p.Arg347Ala ABCC7 p.Arg347Ala ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala ABCC7 p.Ala349Ser ABCC7 p.Ala349Ser ABCC7 p.Ala349Ser ABCC7 p.Cys343Ala ABCC7 p.Cys343Ala ABCC7 p.Cys343Ala ABCC7 p.Phe342Ala ABCC7 p.Phe342Ala ABCC7 p.Phe342Ala ABCC7 p.Met348Ala ABCC7 p.Met348Ala ABCC7 p.Met348Ala ABCC7 p.Val350Ala ABCC7 p.Val350Ala ABCC7 p.Val350Ala ABCC7 p.Val345Ala ABCC7 p.Val345Ala ABCC7 p.Val345Ala ABCC7 p.Gln353Ala ABCC7 p.Gln353Ala ABCC7 p.Gln353Ala ABCC7 p.Leu346Ala ABCC7 p.Leu346Ala ABCC7 p.Leu346Ala 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 ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ala ABCC7 p.Arg334Ala ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala 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 ABCC7 p.Arg334Ala 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 ABCC7 p.Asn1138Ala ABCC7 p.Thr1134Ala ABCC7 p.Thr1134Ala ABCC7 p.Ser1141Ala ABCC7 p.Ser1141Ala ABCC7 p.Met1137Ala ABCC7 p.Met1137Ala ABCC7 p.Thr1142Ala ABCC7 p.Thr1142Ala ABCC7 p.Lys335Ala ABCC7 p.Arg334Ala ABCC7 p.Ser1149Ala ABCC7 p.Ser1149Ala ABCC7 p.Val1147Ala ABCC7 p.Val1147Ala ABCC7 p.Asp1152Ala ABCC7 p.Asp1152Ala ABCC7 p.Ile1151Ala ABCC7 p.Ile1151Ala ABCC7 p.Asn1148Ala ABCC7 p.Asn1148Ala ABCC7 p.Ser1150Ala ABCC7 p.Ser1150Ala ABCC7 p.Met1140Ala ABCC7 p.Met1140Ala ABCC7 p.Ile1139Ala ABCC7 p.Ile1139Ala ABCC7 p.Leu1143Ala ABCC7 p.Leu1143Ala ABCC7 p.Leu1135Ala ABCC7 p.Leu1135Ala ABCC7 p.Trp1145Ala ABCC7 p.Trp1145Ala ABCC7 p.Ala1136Ser ABCC7 p.Ala1136Ser ABCC7 p.Gln1144Ala ABCC7 p.Gln1144Ala ABCC7 p.Ala1146Ser ABCC7 p.Ala1146Ser ABCC7 p.Val1153Ala ABCC7 p.Val1153Ala 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 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala ABCC7 p.Thr339Ala ABCC7 p.Thr339Ala ABCC7 p.Lys335Ala ABCC7 p.Lys335Ala ABCC7 p.Arg334Ala ABCC7 p.Arg334Ala ABCC7 p.Ile344Ala ABCC7 p.Ile344Ala ABCC7 p.Thr351Ala ABCC7 p.Thr351Ala ABCC7 p.Ile340Ala ABCC7 p.Ile340Ala ABCC7 p.Ile336Ala ABCC7 p.Ile336Ala ABCC7 p.Arg347Ala ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala ABCC7 p.Ala349Ser ABCC7 p.Ala349Ser ABCC7 p.Cys343Ala ABCC7 p.Cys343Ala ABCC7 p.Phe342Ala ABCC7 p.Phe342Ala ABCC7 p.Met348Ala ABCC7 p.Met348Ala ABCC7 p.Val350Ala ABCC7 p.Val350Ala ABCC7 p.Val345Ala ABCC7 p.Val345Ala ABCC7 p.Gln353Ala ABCC7 p.Gln353Ala ABCC7 p.Leu346Ala ABCC7 p.Leu346Ala 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 ABCC7 p.Arg334Ala 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 ABCC7 p.Val350Ala 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 ABCC7 p.Val350Ala 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 ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala 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 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 ABCC7 p.Phe337Ala 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 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 ABCC7 p.Val350Ala 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 ABCC7 p.Val350Ala 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 ABCC7 p.Val1147Ala ABCC7 p.Ile1151Ala ABCC7 p.Asn1148Ala ABCC7 p.Ser1150Ala ABCC7 p.Gln1144Ala 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 ABCC7 p.Phe337Cys ABCC7 p.Phe337Glu 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 ABCC7 p.Phe337Ala Both mutations S341A and F337A significantly decreased single-channel conductance (Fig. 9 and Ref. [29]). Login to comment 185 ABCC7 p.Ser341Ala ABCC7 p.Phe337Ala 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 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 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 ABCC7 p.Ser1141Ala 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 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala ABCC7 p.Thr339Ala ABCC7 p.Thr339Ala ABCC7 p.Lys335Ala ABCC7 p.Lys335Ala ABCC7 p.Arg334Ala ABCC7 p.Arg334Ala ABCC7 p.Ile344Ala ABCC7 p.Ile344Ala ABCC7 p.Thr351Ala ABCC7 p.Thr351Ala ABCC7 p.Ile340Ala ABCC7 p.Ile340Ala ABCC7 p.Ile336Ala ABCC7 p.Ile336Ala ABCC7 p.Arg347Ala ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala ABCC7 p.Arg352Ala ABCC7 p.Ala349Ser ABCC7 p.Ala349Ser ABCC7 p.Cys343Ala ABCC7 p.Cys343Ala ABCC7 p.Phe342Ala ABCC7 p.Phe342Ala ABCC7 p.Met348Ala ABCC7 p.Met348Ala ABCC7 p.Val350Ala ABCC7 p.Val350Ala ABCC7 p.Val345Ala ABCC7 p.Val345Ala ABCC7 p.Gln353Ala ABCC7 p.Gln353Ala ABCC7 p.Leu346Ala ABCC7 p.Leu346Ala 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 ABCC7 p.Phe337Ala ABCC7 p.Met348Ala ABCC7 p.Val350Ala 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 ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ala 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 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 ABCC7 p.Gln353Ala 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 ABCC7 p.Arg334Ala 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 ABCC7 p.Arg352Ala 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 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 ABCC7 p.Thr1142Ala 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 ABCC7 p.Thr1142Ala 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 ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ala (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 ABCC7 p.Phe337Ala 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 ABCC7 p.Phe337Ala ABCC7 p.Thr1134Ala ABCC7 p.Thr1134Ala ABCC7 p.Ser1141Ala ABCC7 p.Ser1141Ala ABCC7 p.Lys335Ala ABCC7 p.Lys335Ala ABCC7 p.Met348Ala ABCC7 p.Met348Ala ABCC7 p.Asp1152Ala ABCC7 p.Asp1152Ala 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 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