Abstract
The mitochondrial KATP channel (mitoKATP) is highly sensitive to ATP, which inhibits K+ flux with K1/2 values of 20-40 microM. This raises the question, how can mitoKATP be opened in the presence of physiological concentrations of ATP? We measured K+ flux in liposomes reconstituted with purified mitoKATP and found that guanine nucleotides are potent activators of this channel. ATP-inhibited K+ flux was completely reactivated by both GTP (K1/2 = 7 microM) and GDP (K1/2 = 140 microM). These ligands had no effect in the absence of ATP. The K1/2 for ATP inhibition exhibited quadratic dependence on [GTP] and [GDP], consistent with two binding sites for guanine nucleotides. We also found that palmitoyl-CoA and oleoyl-CoA inhibited K+ flux through reconstituted mitoKATP with K1/2 values of 260 nM and 80 nM, respectively. This inhibition was reversed by GTP (K1/2 = 232 microM) as well as by the K+ channel openers cromakalim (20 microM) and diazoxide (10 microM). Inhibition of mitoKATP by long-chain acyl-CoA esters, like that of ATP, exhibited an absolute requirement for Mg2+ ions. We propose that the open-closed state of the mitochondrial KATP channel is determined by the relative cytosolic concentrations of GTP and long-chain acyl-CoA esters.
Highlights
Mitochondrial KATP channels1 were first discovered in 1991 [1,2,3]
We made the simple demonstration that ATP inhibited Kϩ uptake to rates similar to those of TEAϩ uptake and had no effect on TEAϩ uptake itself. These studies left us with a conundrum: given the high affinity for ATP, how can mitoKATP ever be opened under normal physiological conditions? We hypothesized [4] that endogenous activators of mitoKATP must exist to overcome the high affinity for ATP, and we present support for this hypothesis
Inhibition of mitoKATP by longchain acyl-CoA esters with high affinity is consistent with a proposed signaling role of this channel in regulating -oxidation of fatty acids [13]
Summary
Mitochondrial KATP channels (mitoKATP)1 were first discovered in 1991 [1,2,3]. Inoue et al [1] reported electrophysiological evidence from patch clamp studies of fused mitoplasts, and we described reconstitution of a highly purified mitoKATP [2, 3]. Kϩ flux through the MgATP-inhibited channel is restored to full activity by GTP and GDP, neither of which has any effect in the absence of MgATP. Additional experiments (not shown) further characterize guanine nucleotide reversal of ATP inhibition of Kϩ flux through mitoKATP.
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