ABCMdb

ABCC7 p.Lys1250Met

None has been submitted yet.

Publications

PMID: 12034762 [PubMed] Dousmanis AG et al: "Distinct Mg(2+)-dependent steps rate limit opening and closing of a single CFTR Cl(-) channel."

No. Sentence Comment

246 Nucleotide binding assays (at 0ЊC to prevent hydrolysis) using 8-azidoATP photolabeling show that binding occurs with the same micromolar apparent affinity in wild-type, mutant K1250M, and double mutant K464A/ K1250A, CFTR (Carson et al., 1995). Login to comment

PMID: 12457238 [PubMed] Ando-Akatsuka Y et al: "Down-regulation of volume-sensitive Cl- channels by CFTR is mediated by the second nucleotide-binding domain."

No. Sentence Comment

6 Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Login to comment
123 To check whether ATP hydrolysis at NBD2 is required for CFTR`s regulatory function, we generated two other NBD2-mutants of CFTR, K1250M and D1370N. Login to comment
125 The plasmalemmal distribution of K1250M and D1370N CFTR was confirmed by immunofluorescence microscopy (Fig. 6A) and immunoblotting (Fig. 6B). Login to comment
131 Relative integrated optical densities of the mature bands are shown as percentages of the respective b-actin bands (taken as 100%) In whole-cell patch-clamp experiments, cells expressing the K1250M or D1370N mutant exhibited VSOR currents (Fig. 7A) as large as those observed in mock-transfected cells (Fig. 3A). Login to comment
137 Since VSOR-non-regulating NBD2 mutants (G1349D, K1250M and D1370N) were expressed to approximately the same level as VSOR-regulating WT and G551D CFTR (as seen from immunostaining and Western blotting data), we may exclude the possibility that the down-regulation is simply a side-effect of overexpression of a foreign protein. Login to comment
155 This was, in fact, confirmed by a yeast two- Fig. 7A, B Effects of expression of Walker A or Walker B mutants on VSOR current densities in HEK293T cells. A Time course of VSOR current activation by hypotonic stimulation of cells transfected with the K1250M (top) or D1370N (bottom) mutant, taken during application of alternating pulses from 0 to €40 mV every 15 s. B VSOR current densities from mock-transfected, K1250M mutant-transfected and D1370N mutant-transfected cells, recorded at +40 mV after reaching a steady-state level. Login to comment
175 In our study, the K1250M and D1370N mutations effectively abolished the down-regulatory effect of CFTR on VSOR currents. Login to comment

PMID: 19837660 [PubMed] Chen JH et al: "Direct sensing of intracellular pH by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel."

No. Sentence Comment

6 Because these data suggest that pHi modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pHi dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR). Login to comment
46 These included (i) mouse mammary epithelial cells (C127 cells) expressing wild-type human CFTR, the CFTR variant ⌬R-S660A (13) or the CF mutant G1349D (14), (ii) Fischer rat thyroid epithelial cells expressing the CF mutant G551D (15), and (iii) NIH-3T3 cells expressing the CFTR construct K1250M (16). Login to comment
229 At pHi 7.3, bursts of K464A-CFTR channel openings were separated by prolonged channel closures, whereas dramatically prolonged bursts of K1250M-CFTR channel openings were separated by very long-lived channel closures (Figs. 7, E and G, and 8). Login to comment
232 By contrast, the pHi sensitivity of K1250M-CFTR differed strikingly from that of wild-type CFTR. Login to comment
233 At pHi 6.3, the Po of K1250M-CFTR was reduced because MBD was decreased 0.7-fold, whereas IBI was unchanged (Figs. 7, G and H, and 8). Login to comment
234 At pHi 8.3, the Po of K1250M-CFTR was increased, albeit slightly, as a result of a small increase in MBD and no change in IBI (Figs. 7H and 8). Login to comment
235 Fig. 7H also reveals that the Po of K1250M-CFTR was similar to that of wild-type CFTR at pHi 8.3, but greatly diminished at pHi 7.3 and 6.3. Login to comment
236 Thus, the pHi sensitivity of K1250M-CFTR is the converse of that of wild-type CFTR. Login to comment
246 A, C, E, and G, representative recordings show the effects of pHi on the activity of G551D-, G1349D-, K464A-, and K1250M-CFTR Cl-channels in the presence of ATP (1 mM). Login to comment
247 Dotted lines indicate where channels are closed, and downward deflections correspond to channel openings. B, D, F, and H, effects of pHi on the NPo of G551Dand G1349D-CFTR and Po of K464A- and K1250M-CFTR. Login to comment
284 Burst analysis of D572N- and K1250M-CFTR. Login to comment
285 A and B, MBD and IBI of D572N- and K1250M-CFTR at different pHi values; wild-type CFTR data are shown for comparison. Login to comment
286 Data are means Ϯ S.E. (D572N- and K1250M-CFTR, n ϭ 3; wild-type-CFTR, n Ն 6). Login to comment
312 By contrast, Hϩ ions are either without effect (D1370N-CFTR) or inhibit (K1250M-CFTR) the gating behavior of site-directed mutations in ATP-binding site 2. Login to comment
343 The pHi Sensitivity of CFTR Reveals Cross-talk between ATP Binding Sites 1 and 2-An interesting aspect of our data is the pHi sensitivity of K1250M-CFTR, which is the reverse of that of wild-type CFTR. Login to comment
345 If K1250M-CFTR severely disrupts the function of site 2 (16), these hidden minor pHi effects might originate from site 1. Login to comment

PMID: 9920885 [PubMed] Lee MG et al: "Regulation of Cl-/ HCO3- exchange by cystic fibrosis transmembrane conductance regulator expressed in NIH 3T3 and HEK 293 cells."

No. Sentence Comment

52 The mutagenesis primers were as follows: P205S primer, 5Ј-CGT GTG GAT CGC TTC TTT GCA AGT GGC-3Ј; W846term, 5Ј-GAG CAT ACC AGC AGT GAC TAC ATA GAA CAC ATA CCT TCG ATA TAT TAC-3Ј; G1247D/G1249E, 5Ј-GTG GGC CTC TTG GGA AGA ACT GAT TCA GAG AAG AGT ACT TTG TTA TCA GC-3Ј; K1250M, 5Ј-CTT GGG AAG AAC TGG ATC AGG GAT GAG TAC TTT GTT ATC AGC-3Ј; D1370N, 5Ј-GTA AGG CGA AGA TCT TGC TGC TTA ATG AAC CCA GTG CTC ATT TGG ATC-3Ј. Login to comment
163 Fig. 6 (i-k) shows the plasma membrane localization of K1250M CFTR, D1370N CFTR, and the double mutant G1247D/ G1249E CFTR, respectively. Login to comment
240 The K1250M CFTR mutant had increased channel activity (32), was expressed in the plasma membrane (Fig. 6i) and activated AE similar to WT CFTR (Fig. 12b). Login to comment
265 The K1250M mutant was at least as effective as WT CFTR in stimulating AE activity (b). Login to comment

PMID: 9922377 [PubMed] Gadsby DC et al: "Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis."

No. Sentence Comment

471 Although this would seem to rulestill able to open CFTR channels with a mutation, K1250M, in the NBD2 Walker A motif that was expected to impair out the postulated close structural relationship between G proteins and CFTR`s NBDs, the assignment of their rolesATP hydrolysis there (3). Login to comment

PMID: 9521779 [PubMed] Urbatsch IL et al: "Mutations in either nucleotide-binding site of P-glycoprotein (Mdr3) prevent vanadate trapping of nucleotide at both sites."

No. Sentence Comment

254 In the cystic fibrosis transmembrane conductance regulator (CFTR), mutations of the conserved Walker A lysine altered the conductive properties of the Cl- channel: the K464A mutation in NB1 decreased the frequency of channel openings, whereas K1250A or K1250M in NB2 prolonged the open state of the channel (59). Login to comment

PMID: 9674722 [PubMed] Schwiebert EM et al: "Cystic fibrosis: a multiple exocrinopathy caused by dysfunctions in a multifunctional transport protein."

No. Sentence Comment

224 In NBD2, a few key mutations have been found that include missense mutations (G1349D, D1370N, K1250M, K1250Q, G1244E, S1255P) and several nonsense mutations (W1282X, S1255X, W1316X). Login to comment

PMID: 9530164 [PubMed] Berger HA et al: "Fluoride stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity."

No. Sentence Comment

178 We found that F- stimulates CFTR-K1250M channels to a greater extent than wild-type CFTR (Fig. 8). Login to comment
221 More strikingly, PPi stimulated less current in K1250M-CFTR than in wild-type CFTR (14), whereas F- stimulated more current in K1250M-CFTR than in wild-type CFTR. Therefore, there are distinct differences between the effects of F- and PPi. Login to comment
230 Effect of increasing F- concentration on current in wild-type CFTR, CFTR-K464A, and CFTR-K1250M. Login to comment
171 We found that F2 stimulates CFTR-K1250M channels to a greater extent than wild-type CFTR (Fig. 8). Login to comment
213 More strikingly, PPi stimulated less current in K1250M-CFTR than in wild-type CFTR (14), whereas F2 stimulated more current in K1250M-CFTR than in wild-type CFTR. Therefore, there are distinct differences between the effects of F2 and PPi. Login to comment
222 Effect of increasing F2 concentration on current in wild-type CFTR, CFTR-K464A, and CFTR-K1250M. Login to comment

PMID: 9346969 [PubMed] Ma J et al: "Function of the R domain in the cystic fibrosis transmembrane conductance regulator chloride channel."

No. Sentence Comment

258 Point mutations within the conserved Walker A motif of NBF1 decreased the opening rate of the CFTR channel, while the corresponding mutations in NBF2 (K1250A, K1250M) prolong the open lifetime of CFTR (14, 15). Login to comment
259 The functional effects of K1250A and K1250M on the CFTR channel are similar to the effects of AMP-PNP and PPi (19, 24), suggesting that a decrease in the ATP hydrolysis rate at NBF2 leads to prolonged opening of the CFTR channel. Login to comment
260 The functional effects of K1250A and K1250M on the CFTR channel are similar to the effects of AMP-PNP and PPi (19, 24), suggesting that a decrease in the ATP hydrolysis rate at NBF2 leads to prolonged opening of the CFTR channel. Login to comment

PMID: 9511929 [PubMed] Devidas S et al: "CFTR: domains, structure, and function."

No. Sentence Comment

113 (1995) demonstrated that CFTR variants which contained mutations in the conserved Walker A motif of either NBD1 (K464A) or NBD2 (K1250M and K1250A) decreased the open probability of the channel compared to wt-CFTR. Login to comment

PMID: 8702904 [PubMed] Cotten JF et al: "Effect of cystic fibrosis-associated mutations in the fourth intracellular loop of cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

148 We found that two NBD1 mutants, K464A and G551S, had a normal or increased response to PPi (Fig. 7C). Login to comment
149 In contrast, mutation of two analogous residues in NBD2, K1250M TABLE I Effect of ICL4 mutations on anion selectivity Channels were activated by PKA and 1 mM ATP. Login to comment

PMID: 8572187 [PubMed] Howard M et al: "Epitope tagging permits cell surface detection of functional CFTR."

No. Sentence Comment

251 M2-901 CFIR 5 0 -1 mV C constructed two dgCFTR mutants that are not associated with disease, K1250M and D1370N, also bearing This is a technical study that identifies sites appropri- the M2 epitope at position 901. Login to comment
255 Cell surface expression of M2-901/CFTR has been observed in a expression of M2-901-labeled G551D, G1349D, K1250M, and D1370N dgCFTRs correlates with carbohydrate variety of cell types including HEp-2, BSC40, and addition (Fig &A-II). Login to comment
265 A: G551D; B: G1349D; C: K1250M; D: D1370N; E: AF508; F: N1303K. Login to comment

PMID: 7543023 [PubMed] Gunderson KL et al: "Conformational states of CFTR associated with channel gating: the role ATP binding and hydrolysis."

No. Sentence Comment

57 These NBF1 and NBF2 mutants, harboring the individual mutations K464A and K1250A, K1250G, K1250M, or K1250T, respectively, were expressed in HEK cells and reconstituted into planar lipid bilayers from which single-channel currents were recorded (Figures 3A and 3B). Login to comment
68 The mean conductance of K1250M channels was 8.9 __. 0.13 (n = 4) pS (Figures 3C and 3D), which is not significantly different from the conductance of the wild-type O1 state. Our data show that the O~--*O= transition is highly asymmetric in wild-type channels, consistent with the large free energy change associated with ATP hydrolysis. Login to comment
70 c KI250G ' C K1250M - :~:-~ ~ . Login to comment
76 (B)Single-channelrecordsof K1250A,K1250G, K1250M, and K1250T filtered at 50 Hz under standard cis bath conditions. Login to comment
241 Acknowledgments Correspondence should be addressed to R. R. K. We thank M. Welsh for kindly providing the K1250M and D1370N mutants, P. Quinton for his insightful discussions and critical reading of the manuscript, C. Login to comment
242 Acknowledgments Correspondence should be addressed to R. R. K. We thank M. Welsh for kindly providing the K1250M and D1370N mutants, P. Quinton for his insightful discussions and critical reading of the manuscript, C. Login to comment

PMID: 7539989 [PubMed] Gadsby DC et al: "The CFTR chloride channel of mammalian heart."

No. Sentence Comment

110 The conclusion is that ATP hydrolysis, most likely at NBD1 (because ATP regulates gating of an NBD2 mutant, K1250M), is required to open CFfR channels (2). Login to comment

PMID: 9137558 [PubMed] Sheppard DN et al: "Inhibition of the cystic fibrosis transmembrane conductance regulator by ATP-sensitive K+ channel regulators."

No. Sentence Comment

68 We studied CFTR-containing mutations that affect each of the three types of domains of CFTR: K3 35E, which contains a mutation in the sixth transmembrane segment; K1250M, which contains a mutation in the second NBD; and CFTRAR, where part of the R domain has been deleted (amino acids 708-835). Login to comment

PMID: 7694298 [PubMed] Smit LS et al: "Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

129 Of particular interest is the observation (28) that mutation of the Walker A lysine in NBF2 to methionine (K1250M) produced a 4-fold reduction in the apparent Kil2 for the activation of CFTR by ATP but also completely eliminated inhibition by 1 mM ADP. Login to comment

PMID: 1281220 [PubMed] Sheppard DN et al: "Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents."

No. Sentence Comment

46 MATERIALS AND METHODS For this study we used NIH 3T3 fibroblasts that had been stably infected with a retrovirus expressing either wild-type human CFTR (Gregory, Cheng, Rich, Marshall, Paul, Hehir, Ostedgaard, Klinger, Welsh, and Smith, 1990) or a CFTR mutant in NBD2 (CFIR-KI250M, in which lysine 1250 was changed to methionine) (Anderson et al., 1991a). Login to comment
161 We therefore examined the effect of glibenclamide, the most potent inhibitor we had identified, on CI- currents generated by several CFFR mutants. We studied 100 • 1992 CFTR containing mutations that affect each of the three types of domains of CFTR: CFFP~R where part of the R domain has been deleted (amino acids 708-835); CVFR-K335E, which contains a mutation in the sixth putative membrane-spanning sequence; and CFTR-K1250M, which contains a mutation in the second NBD. Login to comment
173 Comparison with ATP-dependent transporters suggest that mutations in the second NBD, such as CFTR-K1250M, are likely to abolish or impair the function of SHEPP~LRDANDWELSHK-ATP Channel Regulators Inhibit CFTR NBD2 (Anderson et al., 1991a). Login to comment
174 Nevertheless, CFFR-K1250M produces CI- channels that have cAMP-dependent regulation and biophysical properties that are similar to wild-type CFFR (see Anderson et al., 1991a, and Fig. 7, G and I). Login to comment
175 Inhibition of CFTR-K1250M CI- currents by glibenclamide was similar to that observed with wild-type CYI'R (Fig. 7, H and I). Login to comment
176 +SO mV 0 m V ~ -go mV CFTFI~ A~ ......~-1 baseline D CFTR-K335E I ii..i.Iii cAMP .... 13, CFTR-K1250M 1cAMP baseline + gllbenclamlde 250 pA L 50 ms C 1500 1000 5OO -500 o1~0 -100 -75 -50 -25 0 V (mV) ---o-- baseline ---4-- gllbenclamlde E) I cAMP + gllbenclamlde / tp----e F 1000 750 5OO -100 -75 -50 -25 0 V (mV) H ) 1000 PAL__ 50 ms I i cAMP + gllbenclamlde --o-- cAMP --e-- gllbanclamlde / _~_ _ 4OOO 3OOO ~q]O0 I000 0 I000 2OOO 30OO mOO -100 -75 -50 -25 0 V (mV) 25 50 25 50 25 50 FIGURE 7. Login to comment
178 Traces are from C127 cells stably expressing CFTP-~R (A-C), HeLa cells transiently expressing CFFR-K335E (D-F), and NIH 3T3 cells stably expressing CFIR-K1250M (G-I). Login to comment
180 CFFRAR currents were recorded in the absence of cAMP, but for CFFR-K335E and CFTR-K1250M currents cAMP agonists were present. Login to comment
185 Data for steady-state current values measured at +50 and -90 mV are shown for wild-type CF-FR (A), CFTRAR (B), CFFR-K335E (C), and CFTR-K1250M (D). Login to comment
186 The dose-response curves for wild-type CFFR, CFTR-K335E, and CFTR-K1250M were similar. Login to comment
187 Although the effect was not marked, in each case inhibition was slightly more potent at -90 mV than at +50 inV. A 100 8O O 60 c g 20 B 100 8O o 6o eo o m 20 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992 C CFTR 100 ' --o-- 50mV 80 e 6o C g,m 2O 0 20 40 60 80 1C10 0 gllbenclamlde (,aM) D CFTRAR 100 80 2 60 c i 2O 1 ~ ~ 866o o glil~nclamlde (gM) CFTR-K335E 0 20 40 ~ 80 100 glibenclamlda (,ttM) CFTR-K1250M "il o 2i~ ,o 6i) 8o lOO glibenclamlde (i.tM) FIGURE 8. Login to comment
191 (A) Wild-type CFTR; (B) CFTR~R; (C) CFTR-K335E; (D) CFFR-K1250M. Login to comment
196 Half-maximal inhibition by glibenclamide was similar in all cases, occurring at ~ 20 TABLE I1 Effect of Glibenclamide on CFTR Mutants 4-50 mV -90 mV Mutant Experiments Ki n Ki n my/ u2v/ CFTR 21.8 ± 5.2 0.8 - 0.1 19.0 - 5.6* 0.7 ± 0.1 7 CFTR-K335E 25.9 ± 4.1 0.9 ± 0.I 20.4 +--3.7* 0.9 ± 0.1 6 CFTR-K1250M 16.8 _ 4.2 0.8 ± 0.1 31.2 ± 2.5 1.0 - 0.1 5 Ki and n values were calculated as described in Table I for currents measured at +50 and -90 mV for the number of experiments listed. Login to comment
199 Half-maximal inhibition by glibenclamide, measured at -90 mV, showed a small but statistically significant increase in potency compared with that at +50 mV for wild-type CFTR and CFTR-K335E, but not CFFR-K1250M. Login to comment
248 Inhibition of CFTR-K335E and CFTR-K1250M CI- currents by glibenclamide resembled that of wild-type CFFR. Login to comment
249 This suggests that residues K335E and K1250M do not form a critical part of the glibenclamide interaction site. Login to comment

PMID: 1699669 [PubMed] Cheng SH et al: "Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis."

No. Sentence Comment

112 We also made the equivalent mutation within the second nucleotide binding domain (K1250M), and we changed to glutamine both asparagine residues (at 894 and 900) to which carbohydrate is predicted to be attached (N894,900Q) (Riordan et al., 1989). Login to comment
117 Analysis of Mutant Forms of CFTR Expression vectors containing wild-type CFTR (pMT-CFTR, lane 2) and those containing the mutants pMT-CFTR-K464M (lane 3) pMT-CFTR-K1250M (lane 4) pMT-CFTR-Al507 (lane 5) pMT-CFTR-N894. Login to comment
137 CFTR Mutants Mutant CF Exon CFTR Domain A B C Wild type R334W K464M Al507 AF508 F508R s5491 G551 D N894,900Q K1250M Tthllll Y 7 N 9 Y 10 Y 10 N 10 Y 11 Y 11 N 15 N 20 N 22 TM6 NBDl NBDl NBDl NBDl NBDl NBDl ECD4 NBD2 Term - + ++ - + ++ - + - - + - - + - - + - - + - - + ++ + - - - + ++ - + - The known association with CF (Y, yes; N, no), exon localization, domain location, and presence (+ ) or absence (- ) of bands A, B, and C of mutant CFTR species is shown. Login to comment
177 K484M does not produce mature CFTR, whereas K1250M does. Login to comment
179 We need to study the ability of these mutants to complement the chloride channel defect in CF epithelial cells to establish, for example, whether K1250M and K464M both abolish function. Login to comment