Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry

Abstract

Pancreatic ATP-sensitive K + channels (KATP) comprise four inward rectifier subunits (Kir6.2), each associated with a sulphonylurea receptor (SUR1). ATP/ADP binding to Kir6.2 shuts KATP.Mg-nucleotide binding to SUR1 stimulates KATP. In the absence of Mg 2+ , SUR1 increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We simultaneously measured channel currents and nucleotide binding to Kir6.2. Fits to combined data sets suggest that KATP closes with only one nucleotide molecule bound. A Kir6.2 mutation (C166S) that increases channel activity did not affect nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. Mutations at position K205 in SUR1 affected both nucleotide affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in directly contributing to nucleotide binding and in stabilising the nucleotidebound closed state.

Ligand-mediated structural dynamics of a mammalian pancreatic KATP channel

“…Another unique feature of Kir6.2 channels is the requirement of SUR1 co-assembly to achieve the high ATP and open probability of native K ATP channels (Inagaki et al, 1995; Tucker et al, 1997). Several studies have now provided functional, biochemical and structural evidence that SUR1 directly participates in ATP binding via K205 in the L0 linker (Ding et al, 2019; Pratt et al, 2012; Usher et al, 2020). In addition, our structures suggest SUR1 via its TMD0 ICLs and L0 also form a network interaction with Kir6.2 to stabilize it in the CTD-up position to enhance ATP sensitivity.…”

The dynamic interplay of PIP2 and ATP in the regulation of the KATP channel

“…To test this hypothesis, we examined the functional effect of mutations at K39 in two ways; by measuring the ability of ATP to inhibit the KATP current and by exploring ATP binding using a FRET-based assay with fluorescent trinitrophenyl (TNP)-ATP as a congener for ATP 33,34 . The latter method utilises FRET between channels labelled with the fluorescent unnatural amino acid 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) at residue W311 of Kir6.2 and TNP-ATP.…”

“…The latter method utilises FRET between channels labelled with the fluorescent unnatural amino acid 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) at residue W311 of Kir6.2 and TNP-ATP. Incorporation of ANAP into Kir6.2 was achieved as described previously [33][34][35][36] . A GFP tag was also added at the C-terminus of Kir6.2 to facilitate identification of transfected cells.…”

“…Because measurement of ATP binding requires the use of fluorescent TNP-ATP, we next measured the effects of the five mutations introduced into Kir6.2 on TNP-ATP inhibition of the channel (Figure 5A-D). TNP-ATP is a more potent inhibitor of the KATP channel than ATP 33,34 , reducing the IC50 from 34 µM to 1.4 µM (Supplementary table 4). Four of the five mutations (K39A, K39E, E179A, E179K) displayed a similar reduction in current inhibition when tested with TNP-ATP to that found for ATP (Figure 5G, Supplementary table 4).…”

The current chemical biology tool box for studying ion channels

“…Zagotta et al inserted Anap into the N-terminal domain of transient receptor potential cation channel subfamily V member 1 (TRPV1) and labelled the plasma membrane with a metal-chelating lipid to measure protein-membrane distances with transition metal ion Förster resonance energy transfer (Zagotta et al 2016). Puljung et al employed fluorescent nucleotides in combination with Anap-labelled ATP-sensitive K + channel (K ATP ) to study the role of nucleotide binding in the accessory SUR1 subunit (Puljung et al 2019;Usher et al 2020). These studies made use of a double negative eukaryotic release factor that was developed to increase the efficiency of ncAA incorporation and thus fluorescence signals (Schmied et al 2014).…”