Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding

Abstract

The response of ATP-sensitive K+ channels (KATP) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (using FRET with fluorescent nucleotides) of steady-state and time-resolved nucleotide binding to dissect the role of NBS2 of the accessory SUR1 subunit of KATP in channel gating. Binding to NBS2 was Mg2+-independent, but Mg2+ was required to trigger a conformational change in SUR1. Mutation of a lysine (K1384A) in NBS2 that coordinates bound nucleotides increased the EC50 for trinitrophenyl-ADP binding to NBS2, but only in the presence of Mg2+, indicating that this mutation disrupts the ligand-induced conformational change. Comparison of nucleotide-binding with ionic currents suggests a model in which each nucleotide binding event to NBS2 of SUR1 is independent and promotes KATP activation by the same amount.

Photo-Switchable Sulfonylureas Binding to ATP-Sensitive Potassium Channel Reveal the Mechanism of Light-Controlled Insulin Release

“…These forms are strictly correlated with closing and opening of the KATP channel, respectively. 24 , 25 , 27 − 29 Our in silico findings demonstrate that both trans -JB253 and cis -JB253 bind to the same region of SUR1 as the GBM drug does. We show that the light-triggered conformational change of JB253 enhances the probability of SUR1 opening, resulting in an increased probability of KATP closing and insulin release.…”

“…We prefer using these names instead of standard TMD1 and TMD2 since those two legs exhibit a domain swapping between TMD1 and TMD2: H15 and H16 from TMD2 belong to L1 and H9, and H10 from TMD1 belong to L2. Cryo-EM and other experiments 29 show that upon magnesium-adenosine-diphosphate (MgADP) binding to NBD2 and NDB1, both domains stack together, and SUR1 adopts the closed form (outward open). When just one adenosine-triphosphate (ATP) molecule replaces ADP, which happens due to an elevated level of ATP produced by mitochondria after glucose intake, the domains NDB1 and NDB2 dissociate and move apart, and the open SUR1 form (inward open) is observed (see Figure 1 a).…”

Phenotype of a transient neonatal diabetes point mutation (SUR1-R1183W) in mice

“…It is possible that wild-type and mutant channel subunits express in a different ratio in patients and our mice. While it is known that a single mutant Kir6.2 subunit is sufficient to affect the ATP sensitivity of the whole K ATP channel, each SUR1 subunit seems to independently and incrementally contribute to overall channel regulation ( Puljung et al, 2019 ). It is therefore possible that the ratio of mutant to wild-type subunits in our mice is too small to alter the channel ATP sensitivity sufficiently.…”

Genetic Code Expansion: A Brief History and Perspective

“…Shandell, Cornish, and Kass demonstrated the feasibility of sensing the conformational change of a population of UAA-modified cardiac voltage-gated sodium channels expressed in live mammalian cells through incorporation of Anap into the inactivation gate, a dynamic ∼50-amino acid intracellular linker . Since the foundational work of Dougherty and Lester, ion channel physiologists have embraced the UAA technology in oocytes. , Significantly, Puljung and co-workers incorporated Anap into K ATP channels in live mammalian cells, enabling voltage-clamp fluorimetry experiments in this new, possibly more physiologically relevant context . Ligand binding or conformational change measured by Anap environmental sensitivity can now be coupled to functional changes in channel gating measured by electrophysiology in mammalian cells .…”

“…Since the foundational work of Dougherty and Lester, ion channel physiologists have embraced the UAA technology in oocytes. , Significantly, Puljung and co-workers incorporated Anap into K ATP channels in live mammalian cells, enabling voltage-clamp fluorimetry experiments in this new, possibly more physiologically relevant context . Ligand binding or conformational change measured by Anap environmental sensitivity can now be coupled to functional changes in channel gating measured by electrophysiology in mammalian cells . More impactful applications of the technology are being published as technical challenges are overcome and efficiency and ease of use improved.…”