cAMP-dependent Protein Kinase Phosphorylation Produces Interdomain Movement in SUR2B Leading to Activation of the Vascular KATP Channel

Abstract

Vascular ATP-sensitive K ؉ channels are activated by multiple vasodilating hormones and neurotransmitters via PKA. A critical PKA phosphorylation site (Ser-1387) is found in the second nucleotide-binding domain (NBD 2 ) of the SUR2B subunit. To understand how phosphorylation at Ser-1387 leads to changes in channel activity, we modeled the SUR2B using a newly crystallized ABC protein SAV1866. The model showed that Ser-1387 was located on the interface of NBD2 with TMD1 and physically interacted with Tyr-506 in TMD1. A positively charged residue (Arg-1462) in NBD2 was revealed in the close vicinity of Ser-1387. Mutation of either of these three residues abolished PKA-dependent channel activation. Molecular dynamics simulations suggested that Ser-1387, Tyr-506, and Arg-1462 formed a compact triad upon Ser-1387 phosphorylation, leading to reshaping of the NBD2 interface and movements of NBD2 and TMD1. Restriction of the interdomain movements by engineering a disulfide bond between TMD1 and NBD2 prevented the channel activation in a redox-dependent manner. Thus, a channel-gating mechanism is suggested through enhancing the NBD-TMD coupling efficiency following Ser-1387 phosphorylation, which is shared by multiple vasodilators.

Functional Clustering of Mutations in the Dimer Interface of the Nucleotide Binding Folds of the Sulfonylurea Receptor

“…A key feature of the model is that the nucleotide binding sites are located at the dimer interface, such that each nucleotide is interacting with residues from both NBFs. This feature is in agreement with similar NBF homology models of SUR1 (41,42) and SUR2B (43). For clarity, we have defined each ATP-binding site (ABS) as follows: ABS1 is formed by Walker A, Walker B, Q-loop, and H-loop from NBF1, and the Signature Sequence from NBF2, while ABS2 is formed by Walker A, Walker B, Q-loop, and H-loop from NBF2, and the Signature Sequence from NBF1 ( Fig.…”

Phosphorylation-dependent Changes in Nucleotide Binding, Conformation, and Dynamics of the First Nucleotide Binding Domain (NBD1) of the Sulfonylurea Receptor 2B (SUR2B)

“…Phosphorylation Increases the Nucleotide Binding Affinity of NBD1-PKA phosphorylation of SUR2B at NBD1 and NBD2 results in increased K ATP channel activity (24,26). Because K ATP channel gating is also activated by MgATP binding and hydrolysis at the NBDs, we sought to determine whether phosphorylation alters the nucleotide binding properties of NBD1.…”

“…In the case of vascular K ATP channels, monophosphorylation of murine SUR2B at three sites (Thr-633 and Ser-1465 (24) or Ser-1387 (26)) by protein kinase A (PKA) results in K ATP channel activation, with di-phosphorylation of Thr-633 and Ser-1465 further stimulating channel activity (24). Notably, the phosphorylation sites are conserved among species (Thr-635, Ser-1390, and Thr-1547, respectively, in human SUR2B), highlighting their importance in regulating the activity of the SUR NBDs and their subsequent control of K ATP channel function.…”

“…Phosphorylation of SUR2B NBD1, NBD1-⌬C, and N-tail in vitro was achieved by incubating purified proteins with the catalytic subunit of PKA, the in vivo kinase for the SUR proteins (24,26). Deletion of residues Gln-915-Leu-933 from NBD1 to form NBD1-⌬C results in the disappearance of 15 intense resonances centered about 8.2 ppm in the 1 H dimension ( Fig.…”

S-Glutathionylation of Ion Channels: Insights into the Regulation of Channel Functions, Thiol Modification Crosstalk, and Mechanosensing

“…Post-translational modifications (PTMs) are important mechanisms regulating ion channel functions. One of the classical PTM is protein phosphorylation, and a large number of ion channels are found to be regulated by phosphorylation through PKA, PKC, and other protein kinases (30,57,109,112,113,139). A variety of different types of PTMs (e.g., Ubiquitylation, SUMOylation, O-glycosylation/ O-GlcNAcylation, etc.)…”