ABCMdb

PMID: 7589561

Hipper A, Mall M, Greger R, Kunzelmann K
Mutations in the putative pore-forming domain of CFTR do not change anion selectivity of the cAMP activated Cl- conductance.
FEBS Lett. 1995 Nov 6;374(3):312-6., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC7 p.Arg347His ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu ABCC7 p.Arg334His ABCC7 p.Arg334Glu ABCC7 p.Lys335His The following mutations were examined: K335E, R347E, R334E, K335H, R347H, R334H. Login to comment 8 ABCC7 p.Arg347Glu Moreover, anomalous mole fraction behavior for the cAMP activated current could not be detected: neither in wt-CFTR nor in R347E-CFTR. Login to comment 9 ABCC7 p.Arg347His ABCC7 p.Arg334His ABCC7 p.Lys335His Various mutants for which positively charged amino acids were replaced by histidines (K335H, R347H, R334H) did not show pH sensitivity of the IBMX activated wc conductance. Login to comment 20 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu (v) Mutations in the apparent 6th a-helical transmembrane domain of CFTR (K335E, R347E) resulted in changes in the halide selectivity of CFTR [1]. Login to comment 21 ABCC7 p.Arg347Asp (vi) Furthermore, anomalous mole fraction behavior of wt-CFTR was abolished in one mutant of CFTR (R347D) [7]. Login to comment 32 ABCC7 p.Arg347His ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu ABCC7 p.Arg334His ABCC7 p.Arg334Glu ABCC7 p.Lys335His Synthesis of mutated CFTR-cDNA was induced by annealing of ampicillin repair oligonucleotide and oligonucleotide primers carrying the respective mutation changing positively charged to negatively charged amino acids (R334E, R347E, K335E) or replacing R and K at these positions by histidines (R334H, R347H, K335H). Login to comment 76 ABCC7 p.Arg347His ABCC7 p.Lys335Glu ABCC7 p.Lys335His I R334EIIR334HI K335E...... I K335H I R347EII R347H l (n=16) n=10) (n=10) (n=24) (n=9) "° ,11 ml I'lnt;"i' ii Illll 111 0.8 X T °., I ~ 0.4 0.2 I o.o ~ ~ ~ 6!~ 6 ~ 8 ~ ,I I ~ ...... ] J I I L ...... ,j I I t 1 I * *J t........ ~,_J L * * I * *I _ J I .......... I I , * * * , (n=18) (n=lO) (n=22) (n=7) 1.o - T T (n=8) (n=14) T / T T T o.eT T T o 1 "~ 0.4-O 0.2- oo_ L__J , i I i t - - I 1 I I I ~ t J L ' t * J I__~ * I [ * * I l * * j l. Login to comment 80 ABCC7 p.Arg347His ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu ABCC7 p.Arg334His ABCC7 p.Arg334Glu ABCC7 p.Lys335His Next, positively charged amino acids R334, R347, K335 located in the putative 6th pore forming transmembrane a-helical domain of CFTR, were exchanged by histidines (R334H, R347H, K335H) or by the negatively charged glutamate (R334E, R347E, K335E). Login to comment 81 ABCC7 p.Arg347His ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu ABCC7 p.Arg334His ABCC7 p.Arg334Glu ABCC7 p.Lys335His Wc conductances were activated significantly by IBMX in all 6 mutants but to variable degrees (AG in/.tS): 3.2 + 0.6 (R334E, n = 20), 2.7 + 0.6 (R334H, n = 13), 7.1 + 0.9 (K335E, n-- 20), 2.8 + 0.7 (K335H, n = 10), 3.2 + 0.04 (R347E, n = 32) and 1.8 + 0.3 (R347H, n = 10). Login to comment 86 ABCC7 p.Arg347Glu SCN conductance in wt and R347E CFTR and pH &sensitivity of histidine mutants Extracellular C1- (101 mmol/1) was partially (7 mmol/1) or almost completely (96 mmol/l) replaced by equal concentrations of SCN- and we currents were measured in IBMX stimulated oocytes. Login to comment 87 ABCC7 p.Arg347Glu For both wt CFTR and R347E-CFTR we found reduced wc conductance when C1- was replaced by SCN-. Login to comment 88 ABCC7 p.Arg347Glu Anomalous mole fraction behavior could neither be detected for wt-CFTR nor for R347E mutants (Fig. 4). Login to comment 90 ABCC7 p.Arg334Glu It was 2.2 + 0.3 (wt CFTR, n = 17) and 2.0 _+0.2 (R334E, n -- 19), respectively. Login to comment 91 ABCC7 p.Arg347His ABCC7 p.Arg334His ABCC7 p.Lys335His Following previous experiments [7] wc C1- conductances were examined in mutants bearing a histidine mutation (K335H, R347H, R334H) at different extracellular pH values. Login to comment 92 ABCC7 p.Arg347His ABCC7 p.Arg347Glu ABCC7 p.Arg347Glu ABCC7 p.Arg347Glu However, unlike in the previous study in R347H [7] no significant changes of G could be detected when extracellular pH was G wtCFTR R347E 10 - (n=17) 0.8- _T_ T 0~- ~ ~, ~, 0.4- ~ ~ o.2- ~ 0.0-I # 10 0.8 0.60.40.20.0- (n=14) T .__T_ i:I I L J Fig. 4. Summary of the conductance ratios obtained in wt and R347E-CFTR transfected oocytes stimulated by IBMX when 101 mmol/1 extracellular CI- was replaced by (mmol/1) 94 C1- and 7 SCN- (94/7) and 5 C1- and 96 SCN- (5/96), respectively. Login to comment 94 ABCC7 p.Arg347His ABCC7 p.Arg334His ABCC7 p.Arg334His ABCC7 p.Lys335His aL IFEBS Letters 374 (1995) 312-316 - ~ - K335H (n=7) .... ~ .... R347H (n=8) 8 - • R334H (n=5) ...6- I~ ...L 25.5/6 7.5 8/8.5 opH Fig. 5. Summary of the conductances obtained from IBMX stimulated oocytes at different extracellular pH values. Login to comment 95 ABCC7 p.Arg347His ABCC7 p.Arg334His ABCC7 p.Lys335His Experiments were performed with oocytes overexpressing three different CFTR mutants: K335H, R347H, R334H. Login to comment 105 ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu ABCC7 p.Lys95Asp In fact, in a previous study lysine and arginine were replaced by negatively charged amino acids in the first and sixth transmembrane domain (K95D, K335E, R347E), respectively [1]. Login to comment 108 ABCC7 p.Arg347His ABCC7 p.Arg347Glu ABCC7 p.Lys335Glu ABCC7 p.Arg334His ABCC7 p.Arg334Glu ABCC7 p.Lys335His In the present study we repeated some of the published (K335E, R347E, R347H) and performed additional mutations (R334E, R334H, K335H) which are all located in the putative sixth transmembrane domain and overexpressed the respective CFTRs in oocytes. Login to comment 112 ABCC7 p.Arg347Glu ABCC7 p.Arg347Asp ABCC7 p.Lys335Glu Comparable wc measurements were performed in the present study (K335E, R347E compared to R347D in [7]) with SCN- and C1- present in the extracellular bath solution at different concentration ratios. Login to comment 114 ABCC7 p.Arg347Glu Even more important, SCN- con- ductance was not different for the R347E mutant. Login to comment 117 ABCC7 p.Arg347His ABCC7 p.Arg334His ABCC7 p.Lys335His Additional mutations were constructed in which positively charged lysine and two arginines in the sixth transmembrane domain were replaced by pH-sensitive histidines (R334H, K335H, R347H). Login to comment 120 ABCC7 p.Arg347His In the present study, unlike in the previous one ([7], R347H), we did not find significant differences for the cAMP activated wc conductances at different pH values in all three mutants. Login to comment