ABCMdb

PMID: 11600681

Fu J, Ji HL, Naren AP, Kirk KL
A cluster of negative charges at the amino terminal tail of CFTR regulates ATP-dependent channel gating.
J Physiol. 2001 Oct 15;536(Pt 2):459-70., 2001-10-15 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Gly551Asp Other mutations (e.g. G551D) exhibit normal protein trafficking but lower channel activity; many of these mutations are associated with less severe disease (Sheppard et al. 1993; Illek et al. 1999). Login to comment 14 ABCC7 p.Asp58Asn A disease-associated mutant that lacks one of these acidic residues (D58N CFTR) exhibited lower macroscopic currents in Xenopus oocytes and faster deactivation following washout of a cAMP -activating cocktail than wild-type CFTR. Login to comment 16 ABCC7 p.Asp58Asn In excised membrane patches D58N CFTR exhibited a two-fold reduction in single channel open probability due primarily to shortened open channel bursts. Login to comment 18 ABCC7 p.Asp58Ala ABCC7 p.Glu54Ala ABCC7 p.Asp47Ala Replacing this and two nearby acidic residues with alanines (D47A, E54A, D58A) also reduced channel activity, but had negligible effects on bulk PKA phosphorylation or on the ATP dependence of channel activation. Login to comment 23 ABCC7 p.Lys1250Ala Introducing the N-tail mutations into K1250A CFTR, an NBD2 hydrolysis mutant that normally exhibits very long open channel bursts, destabilized the activity of this mutant as evidenced by decreased macroscopic currents and shortened open channel bursts. Login to comment 45 ABCC7 p.Asp58Asn A disease-associated mutant, D58N, maps to one of these acidic residues (Cystic Fibrosis Genetic Analysis Consortium:www.genet.sickkids.on.ca./cftr).Thismutation was found on a patient with CBAVD (Congenital Bilateral Absence of Vas Deferens), a disease that typically associates with mild CF alleles (De Braekeleer & Ferec, 1996). Login to comment 48 ABCC7 p.Asp58Asn Here we report that the D58N CFTR mutant exhibits reduced macroscopic currents and accelerated deactivation kinetics in intact oocytes, and briefer channel openings in excised membrane patches as compared with the wild-type channel. Login to comment 49 ABCC7 p.Asp58Asn The reduced chloride channel activity of D58N CFTR may explain how this mutation causes disease. Login to comment 53 ABCC7 p.Lys1250Ala However, the N-tail mutations prevented the prolonged channel openings that are normally induced by AMP-PNP or by an NBD2 mutation that inhibits ATP hydrolysis (K1250A). Login to comment 58 ABCC7 p.Asp58Ala ABCC7 p.Glu54Ala ABCC7 p.Asp47Ala The N-tail triple mutant (D47A, E54A, D58A) was prepared by using the Stratagene site-directed mutagenesis kit. Login to comment 59 ABCC7 p.Asp58Asn D58N CFTR was constructed by PCR mutagenesis. Login to comment 112 ABCC7 p.Asp58Asn ABCC7 p.Asp58Asn RESULTS D58N CFTR exhibits reduced channel activity The disease-associated D58N CFTR mutant exhibited lower macroscopic currents upon activation with a cocktail containing cAMP, forskolin and IBMX than wild-type CFTR when equivalent cRNA amounts were injected into Xenopus oocytes (Fig. 1). Login to comment 113 ABCC7 p.Asp58Ala ABCC7 p.Glu54Ala ABCC7 p.Asp58Asn ABCC7 p.Asp47Ala The currents mediated by D58N CFTR were somewhat greater than CFTR channel regulation by the amino tailJ. Physiol. 536.2 those observed for a triple mutant in which three acidic residues were replaced with alanines (D47A, E54A, D58A). Login to comment 114 ABCC7 p.Asp58Asn The currents mediated by D58N CFTR and the N-tail triple mutant also deactivated faster than those of wild-type CFTR after removal of the cAMP-containing cocktail (Fig. 1C). Login to comment 117 ABCC7 p.Asp58Asn ABCC7 p.Asp58Asn This finding, coupled with our observation that each of these N-tail mutants can be expressed as the mature form of CFTR with no obvious defect in biosynthetic processing (Naren et al. 1999; Fig. 3 and, for D58N CFTR, unpublished data), suggested to us the possibility that the D58N mutation influences the gating properties of the CFTR channel. Login to comment 119 ABCC7 p.Asp58Ala ABCC7 p.Glu54Ala ABCC7 p.Asp58Asn ABCC7 p.Asp47Ala D58N CFTR and N-tail triple mutant (D47A, E54A, D58A) exhibit lower macroscopic currents and faster deactivation than wild-type CFTR A, schematic diagram of CFTR topology (left) and helical wheel plot of N-tail region of interest (right) showing locations of the mutations analysed in this study. Login to comment 120 ABCC7 p.Asp58Asn B, macroscopic currents for wild-type (WT) CFTR, D58N CFTR and the triple mutant. Login to comment 122 ABCC7 p.Asp58Asn Equal amounts of wild-type, triple mutant or D58N CFTR cRNAs (1 ng) were injected into oocytes. Login to comment 126 ABCC7 p.Asp58Asn Shown are representative current traces and mean estimates of the time to half-maximal deactivation (t1/2; n = 4 for wild-type CFTR, n = 4 for triple mutant CFTR, n = 5 for D58N CFTR). Login to comment 128 ABCC7 p.Asp58Asn Since smaller currents tend to have faster deactivation rates (authors` unpublished observations), higher amounts of mutant CFTR cRNAs were injected (1 ng, 20 ng and 10 ng for wild-type, N-tail triple and D58N mutant CFTR, respectively) in order to achieve comparable macroscopic currents. Login to comment 131 ABCC7 p.Asp58Asn ABCC7 p.Asp58Asn To test the effects of the D58N mutation on CFTR gating we examined the properties of D58N CFTR channels in excised membrane patches. Login to comment 132 ABCC7 p.Asp58Asn D58N CFTR exhibited a lower single channel open probability (Po) under conditions that maximally activate the wild-type channel (Fig. 2). Login to comment 133 ABCC7 p.Asp58Asn Although the Po of D58N CFTR was substantially reduced as compared with the wild-type channel, it was not as markedly reduced as the macroscopic currents in intact oocytes (see Fig. 1B). Login to comment 134 ABCC7 p.Asp58Asn We cannot rule out the possibility that this quantitative difference is due to some effect of the D58N mutation on channel density in the intact oocyte, although it is just as likely that this difference is due to the different activating conditions used in these two assays. Login to comment 136 ABCC7 p.Asp58Asn The reduction in Po exhibited by D58N CFTR was due in large part to a reduction in the duration of channel openings (i.e. to briefer open channel bursts; see Methods for details). Login to comment 141 ABCC7 p.Asp58Asn Mutants in which multiple acidic residues in the N-tail were replaced with alanine were chosen for these and the following experiments, since these mutants exhibit a greater degree of chloride channel dysfunction than the D58N CFTR mutant (Fig. 1 and Naren et al. 1999). Login to comment 153 ABCC7 p.Asp58Asn ABCC7 p.Asp58Asn D58N CFTR exhibits decreased channel activity in excised membrane patches due in part to shorter channel openings A, representative patch clamp records from inside-out excised patches that contained at least two active wild-type (WT) or D58N CFTR channels. Login to comment 154 ABCC7 p.Asp58Asn B, C and D, mean single channel open probabilities, open channel burst durations and interburst durations, respectively, for wild-type CFTR and D58N CFTR. Login to comment 157 ABCC7 p.Asp58Asn The principal effect of the D58N mutation was to reduce the open channel burst duration. Login to comment 161 ABCC7 p.Asp58Asn The results of this analysis of single channel records are consistent with our previous estimates of mean open channel burst duration for this mutant (Naren et al. 1999) and D58N CFTR (Fig. 2) in which multichannel patches were analysed using the cycle time method (see Methods for details). Login to comment 167 ABCC7 p.Asp58Ala ABCC7 p.Glu54Ala The N-tail mutations have little effect on bulk CFTR phosphorylation A, in vitro phosphorylation of wild-type (WT) CFTR and double (E54A, D58A) and triple N-tail mutants. Login to comment 194 ABCC7 p.Lys1250Ala If the N-tail mutations destabilize the long channel openings that predominate under conditions of reduced hydrolysis by NBD2, then they should also inhibit the very long bursts that are characteristic of K1250A. Login to comment 195 ABCC7 p.Lys1250Ala K1250A CFTR is a NBD2 mutant that lacks a lysine at the Walker A motif that is critical for ATPase activity. Login to comment 197 ABCC7 p.Lys1250Ala We introduced the N-tail triple mutations into K1250A CFTR as another test of whether the N-tail regulates gating by affecting a step prior to ATP hydrolysis (i.e. at NBD2). Login to comment 198 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala The triple/K1250A mutant exhibited lower macroscopic currents (Fig. 6A) and faster deactivation (Fig. 6B) compared with K1250A in voltage clamp studies of intact oocytes. Login to comment 199 ABCC7 p.Lys1250Ala In single channel studies we observed that the very long bursts of K1250A CFTR were disrupted by the N-tail mutations (Fig. 7). Login to comment 200 ABCC7 p.Lys1250Ala The triple/K1250A mutant exhibited a lower single channel open probability due to a marked reduction in open channel burst duration (Fig. 7D). Login to comment 203 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala The N-tail mutations inhibit the macroscopic currents and accelerate the deactivation kinetics of the hydrolysis mutant, K1250A CFTR A, macroscopic currents mediated by K1250A CFTR and triple/K1250A CFTR were measured in intact oocytes as described in Fig. 1 legend. Login to comment 204 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala Equal amounts of K1250A and triple/K1250A cRNAs (1 ng) were injected into oocytes (n = 6 oocytes for K1250A, n = 8 for triple/K1250A). Login to comment 205 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala B, deactivation of K1250A (n = 3) and triple/K1250A (n = 4) currents were monitored following washout of cAMP cocktail as described. Login to comment 208 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala The N-tail mutations disrupt the long open channel bursts of K1250A CFTR A and B, representative records from excised inside-out patches for K1250 and triple/K1250A, respectively (1.5 mM MgATP for each). Login to comment 209 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala C and D, mean single channel Po and burst duration for K1250A (n = 4 patches) and triple/K1250A (n = 5 patches). Login to comment 214 ABCC7 p.Asp58Asn In this study we examined the single channel properties and macroscopic current kinetics of a disease-associated mutant that maps to this region of the N-tail (D58N CFTR). Login to comment 217 ABCC7 p.Asp58Asn Our results indicate that: (i) the negative charge at this position is likely to be an important determinant of the involvement of the N-tail in regulating the duration of channel openings and (ii) the mild disease that associates with the D58N mutation could well be due to the partial loss of channel activity that is exhibited by this mutant. Login to comment 220 ABCC7 p.Lys1250Ala However, there does appear to be a rough correlation between macroscopic deactivation kinetics and single channel burst duration for a number of CFTR mutants that have been assayed for both parameters (e.g. K1250A CFTR, which exhibits both prolonged channel bursts and slower macroscopic deactivation; Carson et al. 1995; Gunderson & Kopito, 1995; Wilkinson et al. 1996). Login to comment 222 ABCC7 p.Lys1250Ala If so, perhaps mutations that destabilize (e.g. N-tail mutations) or stabilize (e.g. K1250A) channel openings also influence the stability of channel phosphorylation. Login to comment 228 ABCC7 p.Asp58Ala ABCC7 p.Glu54Ala ABCC7 p.Asp47Ala To further examine the mechanism by which the N-tail modulates ATP-dependent channel gating, we tested the responses of the wild-type and N-tail triple mutant (D47A, E54A, D58A) to ATP and AMP-PNP in single channel studies. Login to comment 231 ABCC7 p.Lys1250Ala In addition, the N-tail mutations destabilized the long bursts that are a feature of an NBD2 mutant (K1250A) that is defective at ATP hydrolysis (Ramjeesingh et al. 1999). Login to comment 241 ABCC7 p.Lys1250Ala We place this transition upstream of ATP hydrolysis because these mutants do inhibit the long bursts that are otherwise activated by manoeuvres that inhibit ATP hydrolysis (AMP-PNP and K1250A mutation). Login to comment 243 ABCC7 p.Lys1250Ala It appears from our data that the N-tail both facilitates the entry of the channel into the longer open state (as evidenced by a decreased rate of activation of N-tail mutants by AMP-PNP) and inhibits exit from this longer open state (as evidenced by destabilization of the long bursts of K1250A CFTR by introducing the N-tail mutations). Login to comment 244 ABCC7 p.Lys1250Ala The results of our K1250A and AMP-PNP experiments appear to link functionally NBD2 and the N-tail in the regulation of CFTR channel gating. Login to comment 255 ABCC7 p.Asp58Asn In this regard, the longer interburst duration exhibited by D58N CFTR (Fig. 2D) implies that this mutation may also inhibit channel opening. Login to comment 260 ABCC7 p.Asp58Asn In addition, our analysis of the D58N CFTR mutant has revealed that a disease-associated mutation that maps to this cluster of acidic residues also inhibits channel activity. Login to comment 270 ABCC7 p.Lys1250Ala AMP-PNP and the K1250A mutation normally prolong CFTR openings by blocking ATP hydrolysis, presumably at NBD2. Login to comment 323 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. Login to comment 384 ABCC7 p.Lys1250Ala They also thank Drs Dale Benos and Michael Quick for their support and guidance, and Dr David Dawson for the K1250A mutant. Login to comment