Reinterpreting the Action of ATP Analogs on KATP Channels

Ulrich Quast,Lindsay Gossack,Joseph Bryan,David Ortiz

doi:10.1074/jbc.m113.476887

Abstract

Neuroendocrine-type K(ATP) channels, (SUR1/Kir6.2)4, couple the transmembrane flux of K(+), and thus membrane potential, with cellular metabolism in various cell types including insulin-secreting β-cells. Mutant channels with reduced activity are a cause of congenital hyperinsulinism, whereas hyperactive channels are a cause of neonatal diabetes. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, thus coupling enzymatic and channel activities. Alterations in SUR1 ATPase activity are proposed to contribute to neonatal diabetes and type 2 diabetes risk. The regulatory model is partly based on the reduced ability of ATP analogs such as adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP) and adenosine 5'-O-(thiotriphosphate) (ATPγS) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR(1E1507Q), with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATPγS, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5'-(β,γ-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[(32)P]ATP from the noncanonical NBD1 of SUR1. This is consistent with structural data on an asymmetric bacterial ABC protein that shows that AMP-PNP binds selectively to the noncanonical NBD to prevent conformational switching. The results imply that MgAMP-PNP and MgAMP-PCP (AMP-PxP) fail to activate K(ATP) channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity.

Highlights

Stimulation by post-hydrolytic, ADP-bound conformations of SUR1 underlies current models of KATP channel activation; ATP analogs are assumed to lower activity by reducing hydrolysis
We found that SUR1E1507Q binds ATP4Ϫ nearly 150 times more tightly than WT, showing that charge at position 1507 in the conserved Walker B phosphate-binding domain is a significant determinant of nucleotide affinity
We propose that AMP-PxP act on SURs to reduce channel activity by stabilizing an inward-facing conformation, and the actions of AMP-PxP reflect the asymmetry of SURs, rather than inhibition of ATP hydrolysis per se

Summary

Background

Stimulation by post-hydrolytic, ADP-bound conformations of SUR1 underlies current models of KATP channel activation; ATP analogs are assumed to lower activity by reducing hydrolysis. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, coupling enzymatic and channel activities. A current model of KATP channel regulation assumes that a post-hydrolytic, ADP-bound enzymatic intermediate or conformation of SUR1 stimulates channel openings, i.e. that hydrolysis is essential for activation (Ref. 19 and reviewed in (20 –22) This model is based in part on studies demonstrating that nonhydrolyzable ATP analogs such as AMP-PNP and AMP-PCP fail to stimulate channel activity, whereas the slowly hydrolyzable ATP␥S stimulates less efficiently than ATP [23,24,25,26,27,28,29,30].

The abbreviations used are

EXPERIMENTAL PROCEDURES

A G T T2 GT

RESULTS

DISCUSSION