ABCMdb

PMID: 12939393

Basso C, Vergani P, Nairn AC, Gadsby DC
Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating.
J Gen Physiol. 2003 Sep;122(3):333-48., [PubMed]

Sentences

No. Mutations Sentence Comment

46 ABCC7 p.Lys464Ala We have previously described pGEMHE-CFTR, pGEMHE-Flag-3-835 (Chan et al., 2000), and pGEMHE-K464A (Vergani et al., 2003). Login to comment 82 ABCC7 p.Lys464Ala Oocytes were injected with 0.1-10 ng of cRNA transcribed from pGEMHE-CFTR (WT or K464A). Login to comment 231 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala The K464A Mutation Little Influences Binding at 0ЊC, but Diminishes Occlusion at 30ЊC, at Low [Nucleotide] Because the mutation K464A at the Walker A lysine in NBD1 impairs CFTR maturation in mammalian cells (e.g., Aleksandrov et al., 2001), we compared nucleotide interactions with WT and K464A CFTR expressed in oocytes, in which we also compared electrophysiological responses. Login to comment 232 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala Despite loading twice as much membrane protein in the K464A lanes as in the WT lanes (Fig. 8 A, right) to compensate for diminished K464A expression (Fig. 8 A, left), negligible occlusion of [␣32P]8-N3ATP at 30ЊC was observed for K464A CFTR, either with or without Vi. Login to comment 233 ABCC7 p.Lys464Ala In contrast, simple binding of 5 or 50 ␮M [␣32P]8-N3ATP, assayed by photolabeling in oocyte membranes at 0ЊC, seemed little different for Flag-K464A than for Flag-WT CFTR (Fig. 8 B), even though that binding occurs predominantly at NBD1 (Fig. 3, above), the structurally altered catalytic site. Login to comment 234 ABCC7 p.Lys464Ala The fact that opening of K464A CFTR channels is impaired at low ␮M [MgATP] (Vergani et al., 2003) suggests that channel opening and nucleotide occlusion at NBD1 share a common step that occurs after the simple association with nucleotide. Login to comment 235 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala The K464A Mutation Virtually Eliminates the Vi-dependent Slowing of CFTR Channel Closing Unlike the slow decline of roughly half of the WT CFTR current (Fig. 4, above), the macroscopic current flowing through K464A channels activated by PKA, MgATP, and Vi decayed relatively rapidly upon nucleotide withdrawal (Fig. 8 C). Login to comment 236 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala The current relaxation after exposure to Vi had a double exponential time course (smooth fit line, Fig. 8 C, center): most of the current decayed rapidly (␶fast ϭ 0.9 Ϯ 0.1 s, n ϭ 10) with a time constant like that of closing (␶b ϭ 620 Ϯ 58 ms, n ϭ 24; Vergani et al., 2003) of K464A channels exposed to just PKA and 5 mM MgATP, indicating that the majority of K464A CFTR channels did not respond to the presence of Vi. Login to comment 250 ABCC7 p.Lys464Ala K464A CFTR, mutated at the Walker A lysine in NBD1, binds but does not occlude micromolar concentrations of 8-N3ATP, in accord with our conclusion from analyses of channel gating that the mutation lowers the apparent affinity for nucleotide interactions required for the transition to the channel open-burst state. Login to comment 257 ABCC7 p.Lys464Ala Mutation K464A in CFTR impairs occlusion at 30ЊC, but not binding at 0ЊC, at low [nucleotide], and disrupts Vi-induced stabilization of open burst state. Login to comment 258 ABCC7 p.Lys464Ala (A, left) Membranes of oocytes expressing WT or K464A CFTR (without Flag tags) were run on SDS-PAGE gels, transferred to nitrocellulose membranes, and blotted with anti-R-domain antibody. Login to comment 259 ABCC7 p.Lys464Ala WT CFTR was expressed at least twice as well as K464A CFTR in this batch of oocytes (arrow marks mature fully-glycosylated CFTR; lower, sharper band is core-glycosylated CFTR). Login to comment 261 ABCC7 p.Lys464Ala Twice as much membrane was used for K464A samples as for WT samples. Login to comment 262 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala (B, left) Immunoblots of membranes from Flag-WTand Flag-K464A-expressing oocytes (the Flag tags facilitated immunoprecipitation to enhanced signal-to-noise ratio, as membranes were not washed before photocrosslinking) blotted with anti-R-domain antibody as in A. Login to comment 263 ABCC7 p.Lys464Ala The membranes contained about one third more WT than mutant K464A protein. Login to comment 265 ABCC7 p.Lys464Ala Approximately 30% more membrane was used for Flag-K464A samples as for Flag-WT samples. Login to comment 266 ABCC7 p.Lys464Ala (C) Macroscopic current in an oocyte patch containing hundreds of K464A CFTR channels. Login to comment 270 ABCC7 p.Lys464Ala (D and E) Mean fit parameters for the time constant (D) and fractional amplitude (E) of the slow component of current decay after activation by 5 mM MgATP plus 300 nM PKA without (white bars) or with 5 mM Vi (black bars) for WT (n ϭ 19) or K464A (n ϭ 10). Login to comment 299 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala Second, purified NBD1 mutant, K464A, CFTR was reported to hydrolyze MgATP at a maximal rate 10-20-fold lower than that of wild-type CFTR, whereas the equivalent mutation in NBD2, K1250A, essentially abolished hydrolysis (Ramjeesingh et al., 1999). Login to comment 300 ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala Unless the specific activity of that K464A CFTR was greatly underestimated (due to unrecognized nonfunctional K464A protein), if NBD1 is essentially catalytically inactive in intact CFTR as we propose, a possible explanation for the reduced hydrolysis by K464A CFTR is that this mutation somehow impairs MgATP hydrolysis at the NBD2 catalytic site. Login to comment 301 ABCC7 p.Lys464Ala Gating measurements offer no support for this possibility, as the K464A mutation little affects normal CFTR-channel gating (Carson et al., 1995; Gunderson and Kopito, 1995; Powe et al., 2002; Vergani et al., 2003) other than reducing the apparent affinity for MgATP (Vergani et al., 2003). Login to comment 302 ABCC7 p.Lys1250Ala However, the mutation does substantially shorten prolonged (locked) open bursts ascribed to NBD2 catalytic-site occupancy by the equivalent of nonhydrolyzed nucleotide, due to the K1250A mutation or to exposure of wild-type CFTR to MgATP plus nonhydrolyzable ATP analogs (Powe et al., 2002; Vergani et al., 2003) or to MgATP plus Vi (Fig. 4 vs. Fig. 8, C and D). Login to comment 303 ABCC7 p.Lys464Ala Although this destabilization could reflect loss of an allosteric influence of the wild-type NBD1 catalytic site on the NBD2 catalytic site, if transition to the open burst state involves formation of an NBD1/NBD2 dimer with interfacial catalytic sites (Vergani et al., 2003), then the K464A mutation could act locally within the interface to reduce stability of the NBD dimer. Login to comment 314 ABCC7 p.Lys1250Ala In accord with this interpretation, NBD2 does appear to hydrolyze ATP (Aleksandrov et al., 2002; cf. Ramjeesingh et al., 1999) and is where the hydrolysis- abolishing mutation, K1250A, similarly (like Vi) prolongs bursts (Carson et al., 1995; Gunderson and Kopito, 1995; Ramjeesingh et al., 1999; Zeltwanger et al., 1999; Vergani et al., 2003). Login to comment 421 ABCC7 p.Gly551Asp ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation. Login to comment