ABCMdb

PMID: 21796338

Qian F, El Hiani Y, Linsdell P
Functional arrangement of the 12th transmembrane region in the CFTR chloride channel pore based on functional investigation of a cysteine-less CFTR variant.
Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Thr1142Cys ABCC7 p.Ser1141Cys ABCC7 p.Met1140Cys ABCC7 p.Asn1138Cys Both S1141C and T1142C could be modified by intracellular [2-sulfonatoethyl] MTS prior to channel activation; however, N1138C and M1140C, located deeper into the pore from its cytoplasmic end, were modified only after channel activation. Login to comment 34 ABCC7 p.Val510Ala As in our recent study on CFTR-TM6 [9], we have used a human CFTR variant ("cys-less" CFTR) in which all endogenous cysteine residues had been substituted by other amino acids [32] and which also includes a mutation in NBD1 (V510A) to increase protein expression in the cell membrane [19]. Login to comment 59 ABCC7 p.Thr1142Cys ABCC7 p.Asn1138Cys a Example time courses of macroscopic currents (measured at -80 mV) carried by N1138C and T1142C as indicated in inside-out membrane patches. Login to comment 62 ABCC7 p.Thr1142Cys ABCC7 p.Asn1138Cys ABCC7 p.Val1147Cys ABCC7 p.Asn1148Cys b Example leak subtracted I-V relationships for N1138C, T1142C, V1147C, and N1148C, recorded from inside out membrane patches following maximal channel activation with PKA, ATP, and PPi. Login to comment 64 ABCC7 p.Thr1142Cys ABCC7 p.Asn1138Cys ABCC7 p.Val1147Cys ABCC7 p.Asn1148Cys As described in the text, whereas MTSES application always led to a decrease in macroscopic current amplitude in reactive mutants, the effects of MTSET were to decrease (e.g., N1138C, V1147C), increase (e.g., T1142C) or not significantly alter (e.g., N1148C) macroscopic current amplitude. Login to comment 80 ABCC7 p.Trp1145Cys ABCC7 p.Thr1142Cys ABCC7 p.Ser1141Cys ABCC7 p.Met1140Cys ABCC7 p.Asn1138Cys ABCC7 p.Val1147Cys ABCC7 p.Asn1148Cys ABCC7 p.Ser1149Cys ABCC7 p.Gln1144Cys Application of MTSES (200 μM) or MTSET (2 mM) to the intracellular solution after channel activation with PKA, ATP, and PPi significantly altered macroscopic current amplitude in nine out of 19 cysteine-substituted mutants tested (N1138C, M1140C, S1141C, T1142C, Q1144C, W1145C, V1147C, N1148C, and S1149C; Figs. 1 and 2). Login to comment 82 ABCC7 p.Ser1141Cys ABCC7 p.Met1140Cys ABCC7 p.Asn1138Cys In the central region of TM12 (N1138C, M1140C, and S1141C), macroscopic current amplitude was decreased by both MTSES and MTSET (Figs. 1 and 2). Login to comment 84 ABCC7 p.Trp1145Cys ABCC7 p.Thr1142Cys ABCC7 p.Gln1144Cys Closer to the intracellular end of TM12 (in T1142C, Q1144C, and W1145C), macroscopic current amplitude was decreased by MTSES application but increased by MTSET (Figs. 1 and 2). Login to comment 86 ABCC7 p.Val1147Cys ABCC7 p.Asn1148Cys ABCC7 p.Ser1149Cys However, unlike previous findings in TM6 [9], we observed a third pattern for cysteines introduced closest to the putative cytoplasmic end of TM12 (V1147C, N1148C, and S1149C). Login to comment 87 ABCC7 p.Val1147Cys ABCC7 p.Asn1148Cys ABCC7 p.Ser1149Cys Here, macroscopic current amplitude was decreased by MTSES application but either decreased (V1147C, S1149C) or not significantly affected (N1148C) by MTSET application. Login to comment 90 ABCC7 p.Leu1143Cys ABCC7 p.Ile1132Cys ABCC7 p.Ile1131Cys ABCC7 p.Ala1136Cys ABCC7 p.Thr1134Cys ABCC7 p.Leu1133Cys ABCC7 p.Ile1139Cys ABCC7 p.Met1137Cys ABCC7 p.Leu1135Cys ABCC7 p.Ala1146Cys A similar lack of effect following prolonged (>5 min) exposure to such high concentrations of both MTSES and MTSET was also observed in ten out of 19 cysteine-substituted mutants tested (I1131C, I1132C, L1133C, T1134C, L1135C, A1136C, M1137C, I1139C, L1143C, and A1146C). Login to comment 92 ABCC7 p.Thr1142Cys ABCC7 p.Ser1141Cys ABCC7 p.Met1140Cys ABCC7 p.Asn1138Cys For MTS reagent-sensitive TM12 mutants located relatively deeply into the pore from its cytoplasmic end (N1138C, M1140C, S1141C, and T1142C), the rate of modification was estimated from the time course of macroscopic current amplitude change following application of MTSES (20-200 μM). Login to comment 93 ABCC7 p.Thr1142Cys ABCC7 p.Ser1141Cys ABCC7 p.Met1140Cys ABCC7 p.Asn1138Cys As shown in Fig. 3a, modification was rapid in M1140C, S1141C, and T1142C, even using a low concentration of MTSES (20 μM), and noticeably slower in N1138C, even with 200 μM MTSES. Login to comment 94 ABCC7 p.Asn1138Cys ABCC7 p.Asn1138Cys The overall pattern of calculated modification rate constants shown in Fig. 3b suggests that modification is faster for cysteines introduced closer to the cytoplasmic end of TM12 and slower at N1138C located further along the axis of TM12. Login to comment 96 ABCC7 p.Ser1141Cys ABCC7 p.Met1140Cys ABCC7 p.Asn1138Cys To gain some information on the orientation of TM12 in the CFTR pore, we therefore examined whether the outermost cysteines introduced into this TM that were sensitive to internal MTS reagents (N1138C, M1140C, and S1141C) could also be modified by extracellular MTSET. Login to comment 101 ABCC7 p.Ser1141Cys ABCC7 p.Met1140Cys ABCC7 p.Asn1138Cys As shown in Fig. 4a, patches excised from MTSET-pretreated cells expressing N1138C, M1140C, or S1141C all gave macroscopic currents that were decreased in amplitude following addition of 2 mM MTSET to the intracellular solution. Login to comment 103 ABCC7 p.Ser1141Cys ABCC7 p.Met1140Cys ABCC7 p.Asn1138Cys These results suggest that none of N1138C, M1140C, or S1141C can be modified covalently by extracellular MTSET. Login to comment 106 ABCC7 p.Thr1142Cys ABCC7 p.Ser1141Cys ABCC7 p.Met1140Cys ABCC7 p.Asn1138Cys We used a similar approach to determine whether N1138C, M1140C, S1141C, and T1142C, located relatively deeply into the pore from its cytoplasmic end and all strongly sensitive to inhibition by intracellular MTSES (Figs. 2 and 3), could be modified by MTSES pretreatment. Login to comment 118 ABCC7 p.Thr1142Cys ABCC7 p.Ser1141Cys Both S1141C and T1142C channels were again rendered insensitive to the test exposure to MTSES, again consistent with them having been covalently modified during pretreatment. Login to comment 119 ABCC7 p.Met1140Cys ABCC7 p.Asn1138Cys In contrast, currents carried by both N1138C and M1140C were strongly inhibited by application of the test dose of MTSES, suggesting that they had not been covalently modified by MTSES pretreatment. Login to comment 120 ABCC7 p.Thr1142Cys ABCC7 p.Ser1141Cys ABCC7 p.Met1140Cys ABCC7 p.Asn1138Cys These results, which are summarized quantitatively in Fig. 5d, suggest that while S1141C and T1142C can be modified by MTSES prior to channel activation, N1138C and M1140C are modified by MTSES only very slowly, if at all, in channels that have not been activated by PKA and ATP. Login to comment 125 ABCC7 p.Ser1141Cys ABCC7 p.Met1140Cys ABCC7 p.Asn1138Cys Extracellular MTSET did not appear able to modify the outermost of these internal MTS-sensitive cysteines (N1138C, M1140C, and S1141C; Fig. 4), consistent with MTS reagents not being permeant. Login to comment 126 ABCC7 p.Ile1132Cys ABCC7 p.Ile1131Cys Previous work from our group suggested that externally applied MTS reagents could modify cysteines only in the outermost part of TM12, namely, I1131C and I1132C [11]; these same cysteines were insensitive to internally applied MTS reagents (Fig. 2), again consistent with impermeability to these reagents. Login to comment 140 ABCC7 p.Thr338Cys ABCC7 p.Thr1142Cys ABCC7 p.Met348Cys ABCC7 p.Lys95Cys ABCC7 p.Ser341Cys ABCC7 p.Val345Cys ABCC7 p.Ile344Cys ABCC7 p.Ser1141Cys ABCC7 p.Pro99Cys ABCC7 p.Met1140Cys ABCC7 p.Asn1138Cys ABCC7 p.Leu102Cys In this respect, the slow rate of modification observed in N1138C (Fig. 3b) is similar to that we reported for P99C and L102C in TM1 [41] and T338C and S341C in TM6 [9], and the much higher modification rate constant for T1142C, S1141C, and (to a lesser extent) M1140C is closer to that reported for K95C in TM1 [41] and I344C, V345C, and M348C in TM6 [9]. Login to comment 143 ABCC7 p.Thr1142Cys ABCC7 p.Ser1141Cys ABCC7 p.Met1140Cys ABCC7 p.Asn1138Cys a Example timecourses of macroscopic current amplitudes (measured at -50 mV) carried by N1138C, M1140C, S1141C, and T1142C as indicated in inside-out membrane patches. Login to comment 148 ABCC7 p.Thr1142Cys ABCC7 p.Ser1141Cys ABCC7 p.Met1140Cys ABCC7 p.Asn1138Cys Using a similar approach, we find that in TM12, S1141C and T1142C can be readily modified by cytoplasmic MTSES prior to channel activation (Fig. 5), whereas N1138C and M1140C are modified rapidly after channel activation (Fig. 3) but very slowly, if at all, prior to channel activation (Fig. 5). Login to comment 151 ABCC7 p.Lys95Cys ABCC7 p.Ser1141Cys Thus, a disulfide bridge can be formed between K95C in TM1 and S1141C in TM12, suggesting that the β carbon distance is in the range of ~5-8 Å for these two introduced cysteines [45]. Login to comment 207 ABCC7 p.Met348Cys ABCC7 p.Val345Cys ABCC7 p.Ala349Cys ABCC7 p.Ile344Cys However, charge-conservative mutations in the analgous part of TM6-for example, in I344C, V345C, M348C, and A349C-also failed to significantly alter Cl-conductance [4]. Login to comment 212 ABCC7 p.Thr1134Phe ABCC7 p.Thr1134Ala ABCC7 p.Met1137Ala In contrast, mutations in the analogous part of TM12 have been found to have little effect on conductance, which was reported as being unaltered in T1134A and M1137A [15] and slightly decreased in the less conservative T1134F [31]. Login to comment