de Wet H et al. (2012), A universally conserved residue in the SUR1 sub...

XB-ART-47258

J Physiol 2012 Oct 15;59020:5025-36. doi: 10.1113/jphysiol.2012.236075.

Show Gene links Show Anatomy links

A universally conserved residue in the SUR1 subunit of the KATP channel is essential for translating nucleotide binding at SUR1 into channel opening.

de Wet H , Shimomura K , Aittoniemi J , Ahmad N , Lafond M , Sansom MS , Ashcroft FM .

???displayArticle.abstract???
The sulphonylurea receptor (SUR1) subunit of the ATP-sensitive potassium (KATP) channel is a member of the ATP-binding cassette (ABC) protein family. Binding of MgADP to nucleotide-binding domain 2 (NBD2) is critical for channel activation.We identified a residue in NBD2 (G1401) that is fully conserved among ABC proteins and whose functional importance is unknown. Homology modelling places G1401 on the outer surface of the protein, distant from the nucleotide-binding site. The ATPase activity of purified SUR1-NBD2-G1410R (bound to maltose-binding protein) was slightly inhibited when compared to the wild-type protein, but its inhibition by MgADP was unchanged, indicating that MgADP binding is not altered. However, MgADP activation of channel activity was abolished. This implies that the G1401R mutation impairs the mechanism by which MgADP binding to NBD2 is translated into opening of the KATP channel pore. The location of G1401 would be consistent with interaction of this residue with the pore-forming Kir6.2 subunit. Channel activity in the presence of MgATP reflects the balance between the stimulatory (at SUR1) and inhibitory (at Kir6.2) effects of nucleotides. Mutant channels were 2.5-fold less sensitive to MgATP inhibition and not activated by MgATP. This suggests that ATP block of the channel is reduced by the SUR1 mutation. Interestingly, this effect was dependent on the functional integrity of the NBDs. These results therefore suggest that SUR1 modulates both nucleotide inhibition and activation of the KATP channel.

???displayArticle.pubmedLink??? 22802590
???displayArticle.pmcLink??? PMC3495298
???displayArticle.link??? J Physiol
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: abcc1 abcc8 cftr kcnj11 mbp tap1

???attribute.lit??? ???displayArticles.show???

	Figure 1. Invariant residues in the NBDs of ABC proteinsA, alignment of the NBDs of various ABC proteins showing the invariant glycine residue that lies C terminal to the Walker A motif. Abbreviations for the eukaryotic proteins are: MRP, multidrug resistance related protein; CFTR, cystic fibrosis transmembrane conductance regulator; PgP, P-glycoprotein; Tap, transporter associated with antigen processing. MsbA, ModC, BtuCD and Sav1866 are bacterial ABC proteins. R, rat; h, human. B and C, homology model of the human SUR1-NBD1 and SUR1-NBD2 dimer (B) and SUR1-NBD2 alone (C). The Walker A motif is shown in red, the Walker B motif in purple, and the ABC signature sequence in orange. G1401 is shown as a sphere. NBD1 is shown in silver (right) and NBD2 in blue (left). The Mg2+ atom is shown as a white sphere and ATP (in ball-and-stick format) in green.
	Figure 2. ATPase activity of NBD2 is decreased by the G1401R mutationATPase activity of isolated MBP-NBD2. Wild-type: circles, n= 4. G1401R: triangles, n= 4. The lines are the best fit of the Michaelis–Menten equation to the mean data.
	Figure 3. ATP inhibition of wild-type and G1401R channels in the absence and presence of Mg2+A and B, KATP currents recorded in response to voltage ramps from −110 to +110 mV in an inside-out patch excised from oocytes expressing wild-type (A) or Kir6.2/SUR1-G1401R (B) channels. The dashed line indicates the zero current level. The bar indicates application of 10 μm ATP. C and D, mean relationships between [ATP] and KATP conductance (G), expressed relative to that in the absence of nucleotide (Gc), for wild-type (•, n= 10), and Kir6.2/SUR1-G1401R (o, n= 8) channels in the absence of Mg2+ (C); or for wild-type (•, n= 8) or Kir6.2/SUR1-G1401R (o, n= 21) currents in the presence of Mg2+ (D). The lines are drawn to eqn (1) with the following parameters. C: wild-type, IC50= 4.7 μm, h= 1.2; G1401R, IC50= 7.3 μm, h= 0.8, a= 0.011. D: wild-type, IC50= 12 μm, h= 1.0; G1401R, IC50= 27 μm, h= 1.1, a= 0.03.
	Figure 4. ADP regulation of wild-type and mutant KATP channelsA, mean wild-type (open bar, n= 8) and G1401R (filled bar, n= 7) currents recorded in the presence of 0.1 mm MgATP, 0.1 mm MgADP or 0.1 mm MgATP plus 0.1 mm MgADP. *P < 0.05. B, mean relationships between [ADP] and KATP conductance (G), expressed relative to that in the absence of nucleotide (Gc), for G1401R channels in the absence (o, n= 5) and presence (•, n= 9) of Mg2+. The lines are drawn to eqn (1) with the following parameters: Mg2+-free, IC50= 49 μm, h= 1.3, a= 0.05; with Mg2+, IC50= 105 μm, h= 1.6, a= 0.09.
	Figure 5. Mg-nucleotide activation of wild-type and mutant KATP channelsA and B, mean relationships between [MgADP] (A) or [MgATP] (B) and KATP conductance (G) expressed relative to that in the absence of nucleotide (Gc), for Kir6.2-G334D/SUR1 (o, n= 5) and Kir6.2-G334D/SUR1-G1401R (•, n= 5) channels. The lines through the G1401R data are drawn by eye. The lines through the wild-type data are drawn to eqn (2) with the following parameters: A: EC50= 38 μm, h= 2, a= 10; B: EC50= 196 μm, h= 1.6, a= 4.6.
	Figure 6. ATP inhibition of G1401R-KAKA channelsMean relationship between [ATP] and KATP conductance (G), expressed relative to that in the absence of nucleotide (Gc), for Kir6.2/SUR1-G1401R-KAKA channels (o, n= 12). The solution contained Mg2+. The line is drawn to eqn (1) with the following parameters: IC50= 9 μm, h= 1.1, a= 0.03. The lines through the data obtained for Kir6.2/SUR1-G1401R in the absence of Mg2+ (dotted line) and Kir6.2/SUR1-G1401R in the presence of Mg2+ (dashed line) are also shown.

References [+] :

Aguilar-Bryan, Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. 1995, Pubmed