ABCMdb

PMID: 9135131

Gribble FM, Tucker SJ, Ashcroft FM
The essential role of the Walker A motifs of SUR1 in K-ATP channel activation by Mg-ADP and diazoxide.
EMBO J. 1997 Mar 17;16(6):1145-52., [PubMed]

Sentences

No. Mutations Sentence Comment

10 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met In addition to its well- residues in the Walker A (WA) motifs of the first known inhibitory effect, Mg-ATP enhances channel (K719A) and second (K1384M) nucleotide-binding activity as evidenced by the fact that when Mg-ATP is domains (NBDs) of SUR1. Login to comment 19 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met ABCC8 p.Lys1384Met When type and K1384M currents, but not K719A or K719A/ Mg2ϩ is present, however, high concentrations of ADP are K1384M currents, suggesting that there may be a inhibitory whereas low concentrations potentiate channel factor in addition to ATP and ADP which regulates activity (Dunne and Petersen, 1986; Kakei et al., 1986; K-ATP channel activity. Login to comment 45 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met ABCC8 p.Lys1384Met We also found that metabolic inhibition led to activation ofγ and β phosphate groups of ATP and is essential for ATP hydrolysis (Azzaria et al., 1989; Saraste et al., 1990; Tian wild-type and K1384M currents, but not K719A or K719A/ K1384M currents. Login to comment 56 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met ABCC8 p.Lys1384Met This may be attributed to relief of the blockingcritical lysine in the WA motifs of either NBD1 (K719A) or NBD2 (K1384M), or both (K719A/K1384M), of SUR1, effect of cytoplasmic ATP (Gribble et al., 1997). Login to comment 59 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met The mean current amplitudes at -100 mV following patch excision were: -3.9 Ϯ 0.8 nAseverely impair nucleotide hydrolysis without significantly affecting nucleotide binding (Azzaria et al., 1989; Saraste (n ϭ 12) for wild-type, -2.2 Ϯ 0.8 nA (n ϭ 12) for K719A, -5.0 Ϯ 1.9 nA (n ϭ 11) for K1384M and -2.6 Ϯ 0.9 nA (n ϭet al., 1990; Tian et al., 1990; Higgins, 1992; Carson et al., 1995; Ko and Pedersen, 1995). Login to comment 60 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met Our results indicate 8) for K719A/K1384M. Login to comment 68 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met ABCC8 p.Lys1384Met Mutant (n ϭ 8) for K719A, 15.7 Ϯ 0.2 µM (n ϭ 6) for K1384M and 16.5 Ϯ 0.3 µM (n ϭ 5) for K719A/K1384M. Login to comment 79 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met Oocytes were coinjected with mutant channels showed 'refreshment` in the presence ofmRNAs encoding Kir6.2 and either wild-type SUR1, K719A-SUR1, Mg2ϩ (Figure 2A), it is unlikely that the ATP hydrolysisK1384M-SUR1 or the double SUR1 mutant K719A/K1384M. Login to comment 81 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met ABCC8 p.Lys1384Met channels (d, n ϭ 10) and the mutant K-ATP channels K719A (j, n ϭ 8), K1384M (u, n ϭ 6) or K719A/K1384M (s, n ϭ 5). Login to comment 86 ABCC8 p.Lys719Ala Solid line fit to wild-type currents; dotted line fit to K719A/ inhibitory. Login to comment 92 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met The Hill coefficients were unaffected by mutation of the WA lysines, being Furthermore, both NBDs are required for channel activation: interaction of Mg-ADP with a single NBD is not1.03 Ϯ 0.06 (n ϭ 10) for currents formed from Kir6.2 and wild-type SUR1, 0.99 Ϯ 0.10 (n ϭ 8) for K719A, sufficient because neither K719A nor K1384M currents were enhanced by Mg-ADP. Login to comment 93 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met ABCC8 p.Lys1384Met In this context, it is of interest1.35 Ϯ 0.13 (n ϭ 6) for K1384M and 0.99 Ϯ 0.14 (n ϭ 5) for K719A/K1384M (n.s., ANOVA). Login to comment 109 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met ABCC8 p.Lys1384Met Figure 6A shows that, K719A-SUR1, K1384M-SUR1 or the double SUR1 mutant K719A/ in the presence of 100 µM Mg-ATP, diazoxide activatedK1384M; 100 µM MgATP and 100 µM MgADP were added to the both wild-type and K1384M currents but did not affectinternal solutions as indicated by the bars. Login to comment 110 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met K719A or K719A/K1384M currents. Login to comment 111 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met When Mg-ATP was reduced to 10 µM, wild-type currents were enhanced by diazoxide, K719A and K719A/K1384M were inhibited(Figure 5). Login to comment 112 ABCC8 p.Lys1384Met The extent of this inhibition was comparable with that observed for wild-type channels when ADP was and K1384M was unaffected (Figure 6B). Login to comment 115 ABCC8 p.Lys1384Met Diazoxide acted more slowly on K1384M than the wild-type channelwere also similar for wild-type and mutant channels. Login to comment 118 ABCC8 p.Lys1384Met The reduced ATP sensitivity of the wild-type channel (Figure 2B) may thus The effects of diazoxide in the presence of the nucleotide diphosphate Mg-ADP are summarized in Figure 6C. Inresult from a simultaneous activation by Mg-ADP (formed from hydrolysis of Mg-ATP) which does not occur in WA the presence of 100 µM Mg-ADP, diazoxide slightly potentiated wild-type and K1384M currents and inhibitedmutant channels. Login to comment 119 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met The fact that the percentage block of the WA mutant currents by Mg-ADP is smaller in the presence K719A and K719A/K1384M currents. Login to comment 123 ABCC8 p.Lys719Ala AMP (100 µM) blocked wild-type currents by 3.9 Ϯ 5.7% (n ϭ 6) and K719A/ that the channel open probability cannot exceed one. Login to comment 130 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met ABCC8 p.Lys1384Met A third(measured between -20 and -100 mV) by 15.3 Ϯ 1.6% (n ϭ 8) in wild-type channels, by 17.4 Ϯ 0.6% (n ϭ 5) possibility, which cannot be completely excluded, is that our ADP solution contains a small quantity of ATP eitherin K719A channels, 18.2 Ϯ 1.0% (n ϭ 4) in K1384M channels and by 12.9 Ϯ 2.3% (n ϭ 6) in K719A/K1384M as a contaminant or formed from ADP by the action by enzymes present in the patch membrane.channels (n.s., ANOVA). Login to comment 136 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met ABCC8 p.Lys1384Met The number of oocytes was: wild type (n ϭ 13), K719A (n ϭ 8), K1384M (n ϭ 12) and K719A/K1384M (n ϭ 5). Login to comment 162 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met ABCC8 p.Lys1384Met Metabolic inhibition increased, and diazoxide further potentiated both wild-type and K1384M whole- ATP sensitivity reported for native K-ATP channels in the absence of Mg2ϩ (Ashcroft and Kakei, 1989).cell currents but was without effect in oocytes expressing K719A or K719A/K1384M. Login to comment 182 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met ABCC8 p.Lys1384Met We Carson et al., 1995; Ko and Pedersen, 1995; Koronakis speculate that nucleotide binding, or hydrolysis, at NBD1 et al., 1995), a similar effect may be expected for K719A, potentiates or prolongs K-ATP channel opening and that K1384M and K719A/K1384M. Login to comment 188 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met A second possibility, therefore, is that to channel activation in intact oocytes, because whole- MgADP hydrolysis at the NBDs might produce some cell K1384M currents, but not K719A currents, were conformational change in SUR1 which enhances K-ATP activated by exposure to azide. Login to comment 189 ABCC8 p.Lys719Ala Since both K719A and channel opening. Login to comment 190 ABCC8 p.Lys1384Met If this idea is correct, then hydrolysis at K1384M channels have similar ATP sensitivities and both NBDs must be required to sustain channel activation neither are upregulated by 100 µM Mg-ADP, this result because mutation of only one NBD removed the ability also argues that, in addition to ATP and ADP, there may of Mg-ADP to activate the current. Login to comment