ABCMdb

PMID: 26209275

Cui G, McCarty NA
Murine and human CFTR exhibit different sensitivities to CFTR potentiators.
Am J Physiol Lung Cell Mol Physiol. 2015 Oct 1;309(7):L687-99. doi: 10.1152/ajplung.00181.2015. Epub 2015 Jul 24., [PubMed]

Sentences

No. Mutations Sentence Comment

110 ABCC7 p.Arg334Cys ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala The amplitude of s1 was b03;25% and s2 was b03;65% of f, which is different from the ratios of s1 and s2 to f in WT-, R334C-, R352A-, and R347A-hCFTR (s1 is b03;40% and s2 is b03;70% of f) (21, 27, 28). Login to comment 113 ABCC7 p.Arg334Cys ABCC7 p.Arg347Ala ABCC7 p.Arg352Ala As we previously reported, R334C-, R347A-, and R352A-hCFTR generally open from the closed state (c), to s1, then opened to s2 and f states (6, 37, 40). Login to comment 119 ABCC7 p.Gly551Asp It has been reported that VX-770 potentiates G551D-hCFTR by increasing open probability and enhancing ATP-independent activity (7, 29, 31). Login to comment 131 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334Ala ABCC7 p.Arg352Ala WT 0.0 0.1 0.2 0.3 Fractional inhibition by 2.5 &#b5;M GlyH-101 # R334C R334A T338A R352A # # # 0.4 0.5 0.4 &#b5;A 50 s ND96 ISO ISO+ GlyH ND96 ISO R334C- hCFTR A B C D ND96 ISO ISO+ GlyH ND96 ISO T338A-hCFTR 1 &#b5;A 50 s 1.0 &#b5;A 50 s ND96 ISO ISO+ GlyH ND96 ISO WT-hCFTR Fig. 5. Login to comment 132 ABCC7 p.Thr338Ala ABCC7 p.Arg334Cys Effects of GlyH-101 on wild-type (WT)- (A), R334C- (B), and T338A-hCFTR (C). Login to comment 155 ABCC7 p.Arg334Ala We have previously reported with the TEVC technique 1.0 &#b5;A 50 s ND96 ISO ISO+ GlyH ND96 ISO WT-mCFTR Fractional inhibition by GlyH 0.0 0.1 0.3 0.4 0.2 0.5 20 40 60 80 100 Concentration (&#b5;M) Kd = 32.39 &#b5;M Fractional activation by GlyH 0.0 0.1 0.3 0.4 0.2 20 40 60 80 100 Concentration (&#b5;M) Kd = 103.56 &#b5;M 0.5 A B C D E Fractional activation by 25 &#b5;M GlyH-101 0.0 0.3 0.1 0.2 F Fractional inhibition by 25 &#b5;M GlyH-101 0.0 0.6 0.2 0.4 # * # 0.5 &#b5;A 50 s R334A-mCFTR ND96 ISO ISO+ GlyH ND96 ISO Fig. 6. Login to comment 162 ABCC7 p.Arg334Ala D: representative current trace of R334A-mCFTR recorded at VM afd; afa;60 mV. Login to comment 163 ABCC7 p.Thr338Ala ABCC7 p.Arg334Ala ABCC7 p.Arg334Ala Extracellular 25 òe;M GlyH-101 failed to block but only potentiated R334A-mCFTR. Summary data for fractional inhibition (E) and fractional potentiation (F) of WT-, V100L-, R147H-, I201V-, R334A-, and T338A-mCFTR with 25 òe;M GlyH-101. Login to comment 167 ABCC7 p.Arg352Ala We first investigated the effect of 2.5 òe;M GlyH-101 on hCFTR with mutations at selected amino acids that are also conserved in mCFTR, selected as follows: 1) R334 sits in the outer mouth of the CFTR pore, attracts Clafa; into the pore, and directly affects ion conduction, which might affect GlyH-101 binding in the pore; 2) T338 is located in the narrow part of the hCFTR pore and has been suggested as a possible binding site for GlyH-101 (21); 3) R352 forms a salt bridge with D993 and D924 and plays a key role in maintaining the open pore architecture of hCFTR (3, 37); consequently, mutation R352A disrupts the R352-D993-D924 salt bridge and may affect the GlyH-101 binding site by altering the hCFTR open pore A B D 800 pA 100 s hCFTR 1 mM ATP+ 127.6 U PKA ATP+PKA+ GlyH-101 control Fractional increase of mCFTR by GlyH-101 1.2 0 20 40 60 80 Kd = 0.60 nM Concentration (nM) 100 0.4 0.8 E 0.4 pA 2 s c f -GlyH-101 2000 # of events 0.0 0.2 -0.2 -0.4 -0.6 -0.8 -1.0 Current (pA) 4000 0.4 pA 2 s c f +GlyH-101 s1 2000 # of events 0.0 0.2 -0.2 -0.4 -0.6 -0.8 -1.0 Current (pA) 4000 6000 NPo 1.0 0 -GlyH +GlyH 2.0 * F 1 nA 100 s mCFTR 1mM ATP+ 127.6 U PKA ATP+PKA+ GlyH-101 control 200 pA 100 s mCFTR 1 mM ATP+ 638 U PKA ATP+PKA+ GlyH-101 control Fig. 7. Login to comment 180 ABCC7 p.Arg334Cys ABCC7 p.Arg334Ala GlyH-101 at 2.5 òe;M blocked WT-hCFTR 28% at VM afd; afa;60 mV in the continuing presence of 10 òe;M ISO (used to activate CFTR via the beta2-adrenergic receptor; see MATERIALS AND METHODS), but the blocking effect was completely lost with both the R334A and R334C mutations (Fig. 5). Login to comment 181 ABCC7 p.Thr338Ala ABCC7 p.Arg352Ala In contrast, 2.5 òe;M GlyH-101 exhibited strengthened block of both T338A- and R352A-hCFTR (Fig. 5, C and D). Login to comment 200 ABCC7 p.Arg334Ala ABCC7 p.Arg334Ala A representative current trace of R334A-mCFTR is shown in Fig. 6D. GlyH-101 completely lost its blocking effect and only exhibited the potentiation function at R334A-mCFTR. Summary data for inhibition are shown in Fig. 6E. GlyH-101 displayed significantly strengthened block of V100L-mCFTR and distinctly weakened block of I201V- mCFTR. Login to comment 300 ABCC7 p.Gly551Asp The VX-770 binding pocket is clearly not generated by the consequences of clinical mutations like G551D because several different channel mutants are sensitive to potentiation by this drug (38). Login to comment 306 ABCC7 p.Arg347His ABCC7 p.Thr338Ile ABCC7 p.Leu1065Pro ABCC7 p.Glu92Lys ABCC7 p.Lys1060Thr ABCC7 p.Glu193Lys ABCC7 p.Arg1066Met ABCC7 p.Ser341Pro However, recent data indicating that VX-770 potentiates channels bearing multiple disease-causing mutations, spread across CFTR, and that VX-770 potentiates one mutant but not another one in the same domain (for example, VX-770 potentiated TM mutants T338I- and R347H- but not S341P- and E92K-CFTR and potentiated cytoplasmic loop mutants E193K- and K1060T- but not R1066M- and L1065P-CFTR) do not support this conclusion (30, 38). Login to comment 311 ABCC7 p.Arg334Ala In fact, we can separate the inhibitory and stimulatory effects of GlyH-101 on mCFTR because the R334A mutation completely abolished the inhibitory effect of GlyH-101 on mCFTR without affecting potentiation. Login to comment