ABCMdb

PMID: 11380256

Gupta J, Evagelidis A, Hanrahan JW, Linsdell P
Asymmetric structure of the cystic fibrosis transmembrane conductance regulator chloride channel pore suggested by mutagenesis of the twelfth transmembrane region.
Biochemistry. 2001 Jun 5;40(22):6620-7., 2001-06-05 [PubMed]

Sentences

No. Mutations Sentence Comment

79 ABCC7 p.Asn1138Ala ABCC7 p.Thr1134Ala ABCC7 p.Ser1141Ala ABCC7 p.Met1137Ala ABCC7 p.Thr1142Ala Five alanine-substitution mutations in TM12 were constructed: T1134A, M1137A, N1138A, S1141A, and T1142A (Figure 1B). Login to comment 81 ABCC7 p.Ser1141Ala Expression of these same mutants in CHO cells yielded similar results (data not shown), with the exception that S1141A expression could not be detected. Login to comment 92 ABCC7 p.Asn1138Ala ABCC7 p.Thr1134Ala ABCC7 p.Ser1141Ala ABCC7 p.Met1137Ala ABCC7 p.Thr1142Ala Block of N1138A (Figure 2B), as well as T1134A, M1137A, and T1142A (data not shown), appeared somewhat weaker than for wild-type, whereas block of S1141A actually appeared stronger than for wild-type (Figure 2B). Login to comment 95 ABCC7 p.Asn1138Ala ABCC7 p.Thr1134Ala ABCC7 p.Met1137Ala ABCC7 p.Thr1142Ala Unitary Current Properties. Expression of wild-type, T1134A, M1137A, N1138A, and T1142A-CFTR in CHO cells led to the appearance of unitary PKAand ATP-dependent Cl-channel currents in excised membrane patches (Figure 3A). Login to comment 96 ABCC7 p.Asn1138Ala ABCC7 p.Thr1134Ala ABCC7 p.Ser1141Ala ABCC7 p.Ser1141Ala ABCC7 p.Ser1141Ala ABCC7 p.Met1137Ala ABCC7 p.Thr1142Ala As described above, S1141A could not be expressed in CHO cells; however, unitary S1141A-CFTR Table 1: Relative Anion Permeabilities for Wild-Type and Mutant CFTRa wild-type T1134A M1137A N1138A S1141A T1142A Cl 1.00 ( 0.01 (10) 1.00 ( 0.06 (5) 1.00 ( 0.03 (6) 1.00 ( 0.02 (4) 1.00 ( 0.02 (5) 1.00 ( 0.04 (7) Br 1.37 ( 0.07 (8) 1.42 ( 0.03 (4) 1.61 ( 0.02 (5)* 1.32 ( 0.08 (7) 1.54 ( 0.05 (4) 1.44 ( 0.04 (5) I 0.83 ( 0.03 (6) 0.85 ( 0.04 (4) 0.88 ( 0.02 (3) 0.83 ( 0.03 (4) 0.78 ( 0.01 (3) 0.86 ( 0.03 (3) F 0.103 ( 0.007 (9) 0.077 ( 0.008 (3) 0.107 ( 0.014 (3) 0.089 ( 0.005 (4) 0.053 ( 0.003 (7)** 0.094 ( 0.007 (3) SCN 3.55 ( 0.26 (7) 3.70 ( 0.33 (4) 3.58 ( 0.14 (5) 3.53 ( 0.21 (6) 3.37 ( 0.35 (3) 3.64 ( 0.22 (5) NO3 1.58 ( 0.04 (10) 1.62 ( 0.05 (4) 1.60 ( 0.04 (3) 1.58 ( 0.05 (6) 1.57 ( 0.03 (3) 1.64 ( 0.07 (3) ClO4 0.25 ( 0.01 (8) 0.22 ( 0.00 (3) 0.29 ( 0.02 (3) 0.44 ( 0.05 (5)** 0.22 ( 0.01 (3) 0.30 ( 0.03 (5) formate 0.24 ( 0.01 (9) 0.26 ( 0.01 (3) 0.27 ( 0.03 (3) 0.27 ( 0.01 (4) 0.26 ( 0.02 (4) 0.25 ( 0.03 (3) acetate 0.091 ( 0.003 (10) 0.102 ( 0.021 (3) 0.103 ( 0.011 (3) 0.087 ( 0.022 (3) 0.066 ( 0.007 (4)* 0.086 ( 0.009 (3) a Relative permeabilities (PX/PCl) for different anions present in the intracellular solution under biionic conditions were calculated from macroscopic current reversal potentials according to eq 1 (see Experimental Procedures), as described in detail previously (16, 20, 36). Login to comment 102 ABCC7 p.Asn1138Ala For each of the five TM12 mutants, the form of the i-V relationship was very similar to that for wild-type (e.g., N1138A; Figure 3B), and the mean slope conductance was not significantly different to that of wild-type (7.8 ( 0.3 pS; n ) 28; Figure 4A). Login to comment 107 ABCC7 p.Asn1138Ala ABCC7 p.Ser1141Ala (B) Mean current remaining following addition of 10 mM SCN- (I/I0) for wild-type (O), N1138A (b, left panel) and S1141A (b, right panel), shown as a function of voltage. Login to comment 109 ABCC7 p.Asn1138Ala ABCC7 p.Thr1134Ala ABCC7 p.Ser1141Ala ABCC7 p.Met1137Ala ABCC7 p.Thr1142Ala Block by SCN- appeared somewhat weakened in N1138A (left), as well as in T1134A, M1137A and T1142A (data not shown, but see Figure 5B), and strengthened in S1141A (right). Login to comment 112 ABCC7 p.Ser1141Ala All unitary currents were recorded in CHO cell patches, except S1141A, which was recorded in BHK cell patches. Login to comment 115 ABCC7 p.Asn1138Ala (B) Mean single channel current-voltage relationship for wild-type (O) and N1138A (b). Login to comment 119 ABCC7 p.Thr1134Ala ABCC7 p.Met1137Ala Under steady-state conditions, each of those mutants which could be expressed in CHO cells appeared to have a lower mean channel open probability (PO) than wild-type, although this difference was only statistically significant for T1134A and M1137A (Figure 4B; P < 0.001, two-tailed t-test). Login to comment 120 ABCC7 p.Ser1141Ala Because of the differences in CFTR channel gating between BHK and CHO cell membrane patches, the PO of S1141A was not measured. Login to comment 126 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser Although no information on the voltage dependence of SCN- block is obtained in this way, we have previously used this same protocol to show directly that intrapore anion binding is altered in the TM6 mutants F337S and T338A (21). Login to comment 128 ABCC7 p.Thr1134Ala ABCC7 p.Ser1141Ala ABCC7 p.Met1137Ala However, the degree of block was significantly reduced in T1134A, M1137A, and S1141A compared to wild-type (Figure 5). Login to comment 136 ABCC7 p.Ser1141Ala (A) Unitary currents carried by wild-type and S1141A-CFTR at -50 mV, with symmetrical 154 mM Cl-containing solutions (control) or after addition of 10 mM NaSCN to the intracellular solution. Login to comment 138 ABCC7 p.Thr1134Ala ABCC7 p.Met1137Ala For wild-type, T1134A, and M1137A, data from these two different methods are in good agreement, however, for other TM12 mutants there is a significant discrepancy [(†) P < 0.01, (‡) P < 0.001, two-tailed t-test]. Login to comment 143 ABCC7 p.Thr1134Ala ABCC7 p.Met1137Ala For wild-type CFTR, as well as for T1134A and M1137A, the inhibitory effects of 10 mM SCN- at -50 mV were the same at the macroscopic and single channel levels (Figure 5B). Login to comment 144 ABCC7 p.Asn1138Ala ABCC7 p.Thr1134Ala ABCC7 p.Ser1141Ala ABCC7 p.Ser1141Ala ABCC7 p.Met1137Ala ABCC7 p.Thr1142Ala However, for N1138A, S1141A, and T1142A, different degrees of inhibition were observed, suggesting that SCN- has some other effect on these mutants which is both distinct from the observed reduction in unitary current amplitude and absent in wild-type CFTR. Since the single channel results report the isolated effects of SCN- on unitary current amplitude, which is presumably determined by the relative tightness of SCN- binding within the pore, we believe that the reduced apparent SCN- binding affinity suggested by the single channel results with T1134A, M1137A, and S1141A is the best reporter of altered pore function in these mutants. Login to comment 156 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala Alanine substitution for these three TM6 residues has been shown to strongly affect conductance, which is greatly reduced in F337A (21) and S341A (13), and significantly increased in T338A (16). Login to comment 158 ABCC7 p.Thr1134Phe Previously, a more dramatic mutation at one of these residues (T1134F) was shown to cause a slight reduction in unitary conductance (13). Login to comment 164 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Asn1138Ala ABCC7 p.Met1137Ala However, as summarized in Table 1, TM12 mutations M1137A and N1138A did not alter the anion selectivity sequence, in stark contrast to the corresponding TM6 mutations F337A (20) and T338A (16). Login to comment 174 ABCC7 p.Thr1134Ala ABCC7 p.Ser1141Ala ABCC7 p.Met1137Ala Block of unitary Cl- currents by SCN- was significantly weakened in T1134A, M1137A, and S1141A compared to wild-type (Figure 5). Login to comment 176 ABCC7 p.Asn1138Ala ABCC7 p.Thr1142Ala This effect did not appear to be a nonspecific result of mutagenesis within TM12, as SCN- block was unaltered in both N1138A and T1142A. Login to comment 177 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser Similar weakening of SCN- block was previously observed in the TM6 mutants F337S and T338A (21). Login to comment 184 ABCC7 p.Thr1134Ala ABCC7 p.Met1137Ala Mean channel PO under steady-state conditions was reduced to 32% of wild-type levels in T1134A, and to 25% of wild-type in M1137A (Figures 3A and 4B). Login to comment 185 ABCC7 p.Asn1138Ala ABCC7 p.Thr1142Ala A similar trend was observed in N1138A and T1142A (Figure 4B), although in these cases the reduction in mean PO was not statistically significant (0.05 < P < 0.15 in both cases, two-tailed t-test). Login to comment 187 ABCC7 p.Ser1118Phe A point mutation in TM11 (S1118F) was recently shown to cause a dramatic reduction in mean channel open burst duration, implying a decrease in the stability of the channel open state (42). Login to comment