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Kir6.2 activation by sulfonylurea receptors: a different mechanism of action for SUR1 and SUR2A subunits via the same residues.
Principalli MA
,
Dupuis JP
,
Moreau CJ
,
Vivaudou M
,
Revilloud J
.
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ATP-sensitive potassium channels (K-ATP channels) play a key role in adjusting the membrane potential to the metabolic state of cells. They result from the unique combination of two proteins: the sulfonylurea receptor (SUR), an ATP-binding cassette (ABC) protein, and the inward rectifier K(+) channel Kir6.2. Both subunits associate to form a heterooctamer (4 SUR/4 Kir6.2). SUR modulates channel gating in response to the binding of nucleotides or drugs and Kir6.2 conducts potassium ions. The activity of K-ATP channels varies with their localization. In pancreatic β-cells, SUR1/Kir6.2 channels are partly active at rest while in cardiomyocytesSUR2A/Kir6.2 channels are mostly closed. This divergence of function could be related to differences in the interaction of SUR1 and SUR2A with Kir6.2. Three residues (E1305, I1310, L1313) located in the linker region between transmembrane domain 2 and nucleotide-binding domain 2 of SUR2A were previously found to be involved in the activation pathway linking binding of openers onto SUR2A and channel opening. To determine the role of the equivalent residues in the SUR1 isoform, we designed chimeras between SUR1 and the ABC transporter multidrug resistance-associated protein 1 (MRP1), and used patch clamp recordings on Xenopus oocytes to assess the functionality of SUR1/MRP1 chimeric K-ATP channels. Our results reveal that the same residues in SUR1 and SUR2A are involved in the functional association with Kir6.2, but they display unexpected side-chain specificities which could account for the contrasted properties of pancreatic and cardiac K-ATP channels.
Figure 1. SUR1 residues Q1342, I1347, and L1350 are essential for K-ATP channel activation by Diazoxide. (A) putative membrane topology of SUR1 (NBD, nucleotide-binding domain; WA, Walker A motif; WB, Walker B motif). The star indicates the region identified by Rainbow et al. (2004) and mutated to obtain the S1M chimera. (B) alignment of the amino acid sequences of human SUR1, rat SUR2A, and human MRP1. Three residues (boxed) are homologous in SURs and not in MRP1: Q1342, I1347, and L1350. Stars and squares indicate the MRP1 residues introduced in SUR1 to obtain SUR1S3M and SUR1S4M, respectively. (C) schematic representation of the chimeras. SUR1 and MRP1 elements are drawn in gray and black, respectively. For clarity, residues Q1342, I1347, and L1350 of SUR1 are indicated by white stripes. The amino acid composition of the constructs were as follows: SUR1S1M = SUR1(M1-V1313) + MRP1(V1261-F1341) + SUR1(R1394-K1582); SUR1S2M = SUR1(M1-P1336) + MRP1(P1284-L1300) + SUR1(V1352-K1582); SUR1S3M = SUR1 with mutations N1338S, I1345V, S1351C, and V1352L; SUR1S4M = SUR1 with mutations K1337S, D1341Q, K1344R, Q1346E, and Q1348R; SUR1(QIL/VFY) = SUR1 with mutations Q1342V, I1347F, and L1350Y, SUR1S2M(VFY/QIL) = SUR1S2M with mutations V1290Q, F1295I, and Y1298L. (D) Diazoxide responses were measured in inside-out patches excised from oocytes coexpressing Kir6.2 and wild type or chimeric SURs or MRP1 as indicated. Diazoxide (300 μmol/L) was applied in the presence of 100 μmol/L ATP, and currents were normalized to the current measured in the absence of nucleotides immediately before opener application. Application of 100 μmol/L ATP alone (black bars) was used as a control. Numbers at right of bars indicate the number of patches included in each average. (E) Representative patch-clamp recordings illustrating the responses of wild type and SUR1(QIL/VFY) channels to 300 μmol/L Diazoxide in the presence of 100 μmol/L ATP. SUR1, sulfonylurea receptor 1; MRP, multidrug-resistance associated protein.
Figure 2. Characterization of the SUR1(QIL/VFY) mutant. (A) Diazoxide dose–response relationships for Kir6.2/SUR1 (circles) and Kir6.2/SUR1(QIL/VFY) (triangles). Currents were normalized to the current measured in 0 ATP. Hill equation fitting yielded K1/2 = 132 μmol/L (h = 1.65) for Kir6.2/SUR1. (B) Response to 100 μmol/L MgADP of SUR1 wt and SUR1(QIL/VFY). Currents were normalized to the current measured in the absence of nucleotides immediately before MgADP application. Numbers indicate the number of patches included in each average. (C) Representative patch-clamp recordings illustrating the responses of wild type and SUR1(QIL/VFY) channels to 100 μmol/L MgADP in the absence of ATP. (D) Current amplitudes in the absence of nucleotides of Kir6.2/SUR1, Kir6.2/SUR1(QIL/VFY), Kir6.2/SUR2A, and Kir6.2/SUR2A(EIL/VFY). (E) ATP dose–response relationships for Kir6.2/SUR1 (circles) and Kir6.2/SUR1(QIL/VFY) (triangles). Currents were normalized to the current measured in 0 ATP. Hill equation fitting yielded K1/2 = 20 μmol/L (h = 1.33) for Kir6.2/SUR1 and K1/2 = 17 μmol/L (h = 1.32) for Kir6.2/SUR1(QIL/VFY). SUR1, sulfonylurea receptor 1.
Figure 3. Openers response by sulfonylurea receptor 1 (SUR1) and SUR2A wt and relative mutants. Residues Q1342, I1347, and L1350 of SUR1, and the matching residues E1305, I1310, and L1313 of SUR2A were mutated into alanine, glycine, and isoleucine. (A) The effects of the application of 100 μmol/L MgADP were measured in inside-out patches excised from oocytes coexpressing Kir6.2 and the indicated SUR constructs. Currents were normalized to the current measured in the absence of nucleotides immediately before MgADP application. Numbers at right of bars indicate the number of patches included in each average. (B) The effects of 300 μmol/L Diazoxide or 10 μmol/L P1075 were measured in inside-out patches excised from oocytes. Diazoxide (300 μmol/L) was applied in the presence of 100 μmol/L ATP. P1075 (10 μmol/L) was applied in the presence of 100 μmol/L ATP. Control designates the current measured in 100 μmol/L ATP before application of the tested opener.
Figure 4. Sulfonylurea receptor 1 (SUR1) homology model based on multidrug resistance protein 1 (Bessadok et al. 2011). (A) Lateral view. The region identified by Rainbow et al. (2004) is highlighted in dark blue (SUR1S1M fragment). Residues Q1342, I1347, and L1350 are represented in ball-and-stick format and colored in red. (B) Close-up lateral view of the cytoplasmic domain. (C) axial view from the extracellular side.
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