ABCMdb

PMID: 21654216

Pratt EB, Shyng SL
ATP activates ATP-sensitive potassium channels composed of mutant sulfonylurea receptor 1 and Kir6.2 with diminished PIP2 sensitivity.
Channels (Austin). 2011 Jul-Aug;5(4):314-9. doi: 10.4161/chan.5.4.16510. Epub 2011 Jul 1., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC8 p.Glu128Lys Recently, we showed that a mutation, E128K, in the N-terminal transmembrane domain of SUR1 disrupts functional coupling between SUR1 and Kir6.2, leading to reduced ATP and PIP2 sensitivities resembling - channels formed by Kir6.2 alone. Login to comment 5 ABCC8 p.Glu128Lys We show here that when E128K SUR1 was coexpressed with Kir6.2 mutants known to disrupt PIP2 gating, the resulting channels were surprisingly stimulated rather than inhibited by ATP. Login to comment 17 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys For two of these residues, R50 and R201, there is evidence that they are involved directly in ATP binding.16 Another mutant, R54E, had WT-like ATP-sensitivity as a single mutant and when it was co-expressed with E128K was still inhibited by 1 mM ATP although the currents were very small (smaller than E128K alone even with 300 bc;M tolbutamide pre-treatment) making it difficult to properly assess ATP sensitivity. Login to comment 18 ABCC8 p.Glu128Lys The R54 residue has previously been implicated in PIP2 affinity or efficacy as mutation of this residue led to decreased channel open probability that could be rectified by exogenous PIP2 .17,18 Two mutants, R177E and R206D, had no detectable currents when co-expressed with either WT SUR1 or E128K SUR1 despite pre-treatment with 300 bc;M tolbutamide. Login to comment 19 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys Strikingly, three Kir6.2 mutants, R176E, R192E and R206E, when coexpressed with E128K SUR1 (denoted as R176E//E128K, R192E//E128K and R206E//E128K hereinafter) displayed increased activity with the application of ATP (Fig. 2A and B). Login to comment 20 ABCC8 p.Glu128Lys The locations of the three Kir6.2 mutations that lead to ATP-activation when combined with E128K SUR1 are shown in a Kir6.2 homology model (Fig. 2C). Login to comment 21 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys All three residues have previously been implicated in PIP2 efficacy.7,8,19-21 Two of them, R176 and R206, lie near the plasma membrane on the cytoplasmic C-terminus of Kir6.2 and may be involved in interactions with the negatively charged phosphate groups in PIP2 .22 The R176E//E128K and R206E//E128K "ATP-activation" mutations showed no or small currents (usually distinguishable single channel openings with conductance consistent with KATP channels) in nucleotide-free Kint/EDTA solution. Login to comment 24 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys N*Po values in control and ATP solutions for R176E// E128K were 0.004 &#b1; 0.002 and 0.029 &#b1; 0.009, respectively, and 0 and 0.306 &#b1; 0.132 for R206E//E128K, respectively (Fig. 2B). Login to comment 25 ABCC8 p.Glu128Lys Exposure of R206E//E128K to 5 bc;M PIP2 also caused a small increase in chosen based upon their placement on the tetrameric Kir6.2 structural model for likelihood to physically interact with E128, which is predicted to lie close to the plasma membrane. Login to comment 29 ABCC8 p.Glu128Lys Next, COSm6 cells were co-transfected with mutant Kir6.2 and either WT or E128K SUR1 plasmids. Login to comment 31 ABCC8 p.Glu128Lys The prediction is that a residue involved in direct electrostatic interaction with E128 of SUR1 would cause significant reduction in ATP-sensitivity when mutated to a negatively charged amino acid and co-expressed with WT SUR1, but would have WT-like ATP sensitivity when co-expressed with E128K SUR1 as the ionic interaction would be re-established. Login to comment 32 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys E128K mutation prevents efficient surface expression of the channel; we have previously shown that this defect is partially corrected by treating cells with a KATP channel antagonist, tolbutamide, which acts as a chemical chaperone.15 Cells expressing the E128K mutation were therefore pre-treated with 300 bc;M tolbutamide overnight to facilitate surface expression. Login to comment 36 ABCC8 p.Glu128Lys Three of the mutations including K47E, R50E and R201E did indeed decrease ATP sensitivity when expressed with WT SUR1; but when co-expressed with E128K, even greater insensitivity to ATP inhibition was observed, indicating the Kir6.2 and SUR1 mutations possibly cause ATP- insensitivity through different, additive channels. Login to comment 38 ABCC8 p.Glu128Lys Recently, we showed that a residue in the TMD0, E128, plays a critical role in the engagement between SUR1 and Kir6.2 by stabilizing Kir6.2 interactions with PIP2 .14 Mutation of this residue to an oppositely charged amino acid, E128K, disrupts functional coupling between the two subunits leading to decreased Po and also decreased PIP2 and ATP sensitivity in full-length channels. Login to comment 39 ABCC8 p.Glu128Lys In mini-KATP channels, E128K also diminishes the Po and bursting behavior conferred by TMD0 and gives rise to an ATP-sensitivity approaching that of Kir6.2ƊC channels. Login to comment 40 ABCC8 p.Glu128Trp Moreover, another mutation at the same site, E128W, causes rapid destabilization of channel activity that is reversed by PIP2 . Login to comment 45 ABCC8 p.Glu128Lys Our simple hypothesis is that E128 interacts electrostatically with a positively charged Kir6.2 residue, which when mutated to a negatively-charged amino acid should suppress the defect brought about by the E128K mutation. Login to comment 46 ABCC8 p.Glu128Lys Although we did not identify a residue that behaved as our hypothesis predicted in this study, we quite unexpectedly identified several mutations within Kir6.2 which, when coexpressed with E128K, were more active in the presence of ATP than in its absence. Login to comment 49 ABCC8 p.Glu128Lys In the presence of Mg2+ , ATP stimulates channel activity by interacting with the nucleotide binding domains of SUR1.24 To exclude the possibility that the low level of stimulation by ATP is due to incomplete chelation of Mg2+ by EDTA, we further examined the effect of ATP on channels formed by one of the Kir6.2 mutants, R176E, and a SUR1 harboring both the subunit to stabilize - channel - activity.23 The R192E//E128K pair usually had current on isolation of the inside-out membrane patch but rapidly inactivated in nucleotide-free Kint/EDTA and only became active again in high concentrations of ATP (1 mM). Login to comment 51 ABCC8 p.Glu128Lys R192E//E128K records were not channel opening (two patches were tested) but the extent of stimulation was far less than that seen with exposure to 5 mM ATP (Fig. 2A, bottom). Login to comment 57 ABCC8 p.Glu128Lys Error bars represent SEM, except R50E//E128K 1 mM ATP which is the difference between the - individual values from the two patches tested; the number of patches tested (n) for each condition is given below the x-axis. Login to comment 58 ABCC8 p.Glu128Lys WT: wild-type; EK: E128K. Login to comment 63 ABCC8 p.Glu128Lys In mutant channels formed by E128K SUR1 and doubly mutant Kir6.2 containing R50E and R176E or R50E and R206E,nostimulatoryorinhibitoryeffects on the low spontaneous channel activity were observed with exposure to 1 mM ATP (data not shown). Login to comment 64 ABCC8 p.Glu128Lys The results suggest that the stimulatory effect of ATP seen in the Kir6.2 R176E or R192E or R206E// E128K SUR1 mutant channels involves the ATP binding site that normally leads to inhibition of channel activity. Login to comment 65 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys E128K mutation and another mutation located in the second NBF, G1479R, that abolishes MgATP stimulation.25 The Kir6.2 R176E//SUR1 E128K-G1479R triple mutant channel was still more active in the presence of 1 mM ATP than in Kint/EDTA (data not shown), indicating that the ATP-activation effect observed is not due to MgATP stimulation via SUR1. Login to comment 68 ABCC8 p.Glu128Lys ATP-activation observed with co-expression of E128K and Kir6.2 mutations that diminish PIP2 response. Login to comment 72 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys (B) Average currents in the absence and presence of ATP were quantified as N*Po values for R176E//E128K (0 vs. 1 mM ATP) and R206E//E128K (0 vs. 5 mM ATP); no activity was seen during control conditions (i.e., no ATP) in any R206E//E128K patch. Login to comment 85 ABCC8 p.Glu128Lys That the E128K mutation in SUR1 causes the channel to become less sensitive to ATP inhibition-as a result of functional uncoupling between SUR1 and Kir6.2-helps unmask the temporary opening of the channel during the brief transitions between ATP-bound and ATP-unbound states. Login to comment 86 ABCC8 p.Glu128Lys ABCC8 p.Glu128Trp The ATP-induced conformational change referred to here is analogous to the previously reported effect of ATP on several mutations that cause spontaneous channel inactivation, including E128W in SUR1 and R192E, R301E and R314E in Kir6.2.14,23 In the inactivation mutants, exposure to high concentrations of ATP followed by subsequent washout to remove the inhibitory effect of ATP recovers channels from inactivation and allows channels to open briefly before they inactivate again (this inactivation phenomenon can be seen in the R192E// E128K mutant following removal of ATP, Fig. 2A, middle). Login to comment 89 ABCC8 p.Glu128Lys In conclusion, the abnormal ATP- stimulation gating behavior we observed in the combined SUR1 E128K and Kir6.2 mutants that have severely impaired ability to interact with PIP2 has provided a glimpse of a gating step associated with ATP binding. Login to comment 91 ABCC8 p.Glu128Lys Proposed mechanism by which ATP stimulates the activity of channels formed by E128K-SUR1 and R176E-, R192E- or R206E-Kir6.2. Login to comment 94 ABCC8 p.Glu128Lys (B) In E128K-SUR1 + WT Kir6.2 channels, functional coupling between SUR1 and Kir6.2 is disrupted causing unstable Kir6.2-PIP2 interactions thereby reduced open probability. Login to comment 96 ABCC8 p.Glu128Lys (C) In channels formed by E128K-SUR1 and Kir6.2 PIP2 mutants, most channels are unable to interact with PIP2 resulting in no channel activity in Kint/EDTA. Login to comment