ABCMdb

PMID: 12457238

Ando-Akatsuka Y, Abdullaev IF, Lee EL, Okada Y, Sabirov RZ
Down-regulation of volume-sensitive Cl- channels by CFTR is mediated by the second nucleotide-binding domain.
Pflugers Arch. 2002 Nov;445(2):177-86. Epub 2002 Sep 7., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Gly551Asp The VSOR regulation by CFTR was not affected by G551D mutation at first nucleotide-binding domain (NBD1), which is known to impair CFTR interaction with the outwardly rectifying chloride channel, ORCC, epithelial amiloride-sensitive Na-channel, ENaC, and renal potassium channel, ROMK2. Login to comment 5 ABCC7 p.Gly1349Asp In contrast, the G1349D mutant, which impairs ATP binding at NBD2, effectively abolished the down-regulatory effect of CFTR. Login to comment 6 ABCC7 p.Asp1370Asn ABCC7 p.Lys1250Met 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 94 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Crucial role of NBD2 in the CFTR-VSOR interaction To clarify the molecular basis for the CFTR-VSOR interaction, we first introduced mutations into NBD1 (G551D), NBD2 (G1349D) and the C-terminal PDZ-binding domain (DTRL). Login to comment 98 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Quantitative densitometry (Fig. 4Bb) revealed that the G551D, G1349D and DTRL mutants were expressed to a comparable extent in the plasma membrane. Login to comment 116 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Gly551 fiAsp and Gly1349 fiAsp (G551D and G1349D) are naturally occurring mutations in NBD1 and NBD2, known to cause a severe CF phenotype by decreasing nucleotide binding at NBDs [25]. Login to comment 117 ABCC7 p.Gly551Asp The G551D mutation has been shown to impair CFTR regulation of ORCC [7, 17] and ENaC [20]. Login to comment 118 ABCC7 p.Gly551Asp In our experiments, however, the G551D mutant was an effective down-regulator of VSOR activity with an efficiency close to that of WT CFTR (Fig. 5). Login to comment 120 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In contrast to the mutation in NBD1, we found that the G1349D mutation failed to affect VSOR currents (Fig. 5), although it was expressed in the plasma membrane to an extent comparable to that of the G551D and DTRL mutants (Fig. 4). Login to comment 123 ABCC7 p.Asp1370Asn ABCC7 p.Lys1250Met 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 ABCC7 p.Asp1370Asn ABCC7 p.Lys1250Met The plasmalemmal distribution of K1250M and D1370N CFTR was confirmed by immunofluorescence microscopy (Fig. 6A) and immunoblotting (Fig. 6B). Login to comment 127 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Fig. 5A, B Effects of expression of WT CFTR and CFTR proteins mutated at NBD1, NBD2 or the PDZ-binding domain on VSOR current densities in HEK293T cells. A Time-course of VSOR current activation by hypotonic stimulation of cells transfected with DTRL (top), G551D (middle) and G1349D (bottom) mutants, taken during application of alternating pulses from 0 to €40 mV every 15 s. B Peak VSOR current densities from HEK293T cells transfected with vector alone (Mock), WT CFTR or one of the three mutants, recorded at +40 mV after reaching a steady-state level. Login to comment 131 ABCC7 p.Asp1370Asn ABCC7 p.Lys1250Met 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 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Asp1370Asn ABCC7 p.Lys1250Met 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 144 ABCC7 p.Gly551Asp The G551D mutant of CFTR, which produces severe lung disease in 3% of all CF patients, is normally transported to the plasma membrane, but does not generate a functional Cl-conductance due to impaired ATP binding at NBD1 [58]. Login to comment 152 ABCC7 p.Gly551Asp As was the case for ORCC (but not for VSOR in the present study), the point mutation at NBD1, G551D, abolished the CFTR-mediated regulation of the epithelial sodium channel in both oocytes [20] and planar lipid membranes [15]. Login to comment 155 ABCC7 p.Asp1370Asn ABCC7 p.Asp1370Asn ABCC7 p.Lys1250Met ABCC7 p.Lys1250Met 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 156 ABCC7 p.Gly551Asp Asterisk, P<0.05 versus mock data which is identical to that presented in Fig. 5B hybrid analysis in which the interaction was sensitive to the G551D mutation [20]. Login to comment 159 ABCC7 p.Gly551Asp The G551D mutation at NBD1, however, abolished the CFTR-dependent component of glibenclamide-induced inhibition. Login to comment 162 ABCC7 p.Gly551Asp Although the features of CFTR-mediated regulation are different in all three cases (activation for ORCC, inhibition for ENaC and glibenclamide sensitivity for ROMK2), the modulation itself is due to a direct or indirect interaction involving NBD1 and is sensitive to the G551D mutation. Login to comment 163 ABCC7 p.Gly551Asp In contrast, the results obtained in the present study show that VSOR differs markedly from these three channels in an important point: CFTR-mediated regulation was insensitive to the CF-causing mutation G551D in NBD1. Login to comment 171 ABCC7 p.Gly1349Asp On the contrary, the mutation G1349D in NBD2, which causes a severe CF phenotype with pancreatic insufficiency, effectively impaired the CFTR-VSOR interaction. Login to comment 175 ABCC7 p.Asp1370Asn ABCC7 p.Lys1250Met In our study, the K1250M and D1370N mutations effectively abolished the down-regulatory effect of CFTR on VSOR currents. Login to comment