A Conserved Inhibitory and Differential Stimulatory Action of Nucleotides on KIR6.0/SUR Complexes Is Essential for Excitation-Metabolism Coupling by KATP Channels

Abstract

The mechanism by which ubiquitous adenine nucleotide-gated K IR 6.0 4 /SUR 4 channels link membrane excitability with cellular metabolism is controversial. Is a decreased sensitivity to inhibitory ATP required, or is the Mg-ADP/ATP-dependent stimulatory action of the ATPase, sulfonylurea receptor (SUR), on K IR sufficient to elicit a physiologically significant open channel probability? To evaluate the roles of nucleotide inhibition versus stimulation, we compared K IR 6.1-based K NDP channels with K IR 6.2-based K ATP channels and all possible K IR 6.1/6.2 hybrids. Although K NDP channels are thought to be poorly sensitive to inhibitory ATP and to require Mg-nucleotide diphosphates for activity, we demonstrate that, like K ATP , and hybrid channels, they are inhibited with an IC 50(ATP) 100-fold lower than The primary signaling mechanism that attunes the P O to the cellular metabolic status remains controversial. Observations that K ATP channels burst spontaneously in inside-out patches and show reversible inhibition by submillimolar ATP (4 -6) suggested that opening these K IR s in vivo, in the face of millimolar nucleotide, would necessitate a significant decrease in their sensitivity to ATP. The reduction in the ATP sensitivity of isolated K ATP channels upon treatment with numerous substances has been interpreted as evidence for the plasticity of ATP-inhibitory gating in vivo. Following the report (7) that ATP weakly inhibits K IR 6.2-based channels in the absence of SUR (IC 50(ATP) ϭ ϳ0.2 mM versus ϳ5-20 M with SURs; Refs. 8 and 9), K IR -based plasticity of ATP inhibition has been considered a possible cause of the variable activity observed for K ATP channel isoforms in situ.An alternative idea is that SURs, obligatory regulatory subunits of K ATP channels and members of the ABC superfamily (10, 11), act as nucleotide sensors and, in vivo, SURs would exert a Mg-ADP/ATP-dependent stimulatory action sufficient to overcome the saturated inhibitory effect of nucleotides at noncanonical nucleotide binding site(s) on the K IR (1). This hypothesis is consistent with only a small fraction, ϳ1%, of the maximal NP O for K ATP channel population participating in the regulation of insulin secretion from pancreatic ␤-cells (12) or shortening of action potentials of metabolically inhibited ventricular cardiomyocytes (13,14). The hypothesis is consistent with the marginal effect of a decreased submembrane concentration of ATP on K ATP channel activity in situ, e.g. the enhancement of K ATP currents observed when adenylyl cyclases are maximally stimulated, thus reducing ATP without producing stimulatory MgADP, in cardiomyocytes dialyzed with submillimolar ATP, but not in the perforated patch or conventional whole-cell configuration with millimolar [ATP] i (15). The physiologic significance of the Mg-nucleotide-dependent stimulation of K IR 6.2 has been established by the identification of SUR1 mutations that compromise the stimulatory, but not the inhibitory action of Mg-nucleotides on pancreatic ␤-cell K ATP channels (...

Toward Linking Structure With Function in ATP-Sensitive K+ Channels

“…The first double mutant, K IR 6.2 Arg50Gln/ Lys185Gln (65), demonstrated the additive contribution of NH 2 -and COOH-terminal residues to ATP-induced stabilization of the closed state, consistent with the possibility that these two residues cooperate in the coordination of separate negatively charged phosphate groups of ATP in SUR1-containing channels. The GIRK1-based homology models of the K IR 6.0 C-domain (13,71) support the idea that Ile182, Lys185, and Gly334 are colocalized and that Lys185 and Gly334 probably contribute to the surface of the adenine-and ␤-phosphate-selective ATP-binding pocket that we have argued is conserved in both K IR 6.0 subunits (39,72). Our modeling of the complete pore indicated an additional key structural aspect of the ATP-inhibitory machinery.…”

“…The effectiveness of this control is supported by the failure of K IR 6.2 subunits to contribute to membrane hyperpolarization in knockout mice lacking SUR1 (30,31). In addition to assembly and trafficking, other studies have emphasized the role of SUR1 in "activating" the pore in the absence of ligands and increasing its sensitivity to inhibitory ATP (32,33); in the stimulatory action of MgADP (34,35); in the action of pharmacologic agents (both openers and inhibitors) (36,37); and, in the case of cardiac and ␤-cell K ATP channels, which share a common K IR 6.2 pore, in determining the isoform differences in slow gating and its modulation by inhibitory and stimulatory nucleotides (38,39 6.0 pores necessary to understand their weak inward rectification and ATP-inhibitory gating has been limited. Eukaryotic ion channels are difficult to express and purify in the quantities needed for structural work.…”

A Novel ABCC8 (SUR1)-dependent Mechanism of Metabolism-Excitation Uncoupling

“…The more significant (p Ͻ 0.005) effect of the mutation at these higher MgATP/ADP ratios indicates that the Q1178R markedly amplifies the stimulatory action of [MgATP] Ͼ 0.5 mM (see later). In 0.5 mM MgADP without ATP, or in 5 mM MgUDP, which subsaturates the stimulatory, but not adenine-selective, inhibitory sites (5,14), both channels display Ͼ90% of the ligand-independent activity, P omax (three pairs of records not shown). Altogether, these observations argue that the hyperactivity of the NDSUR1 channel in physiological [ATP]/[ADP] is predominantly determined by MgATP, regardless of whether the magnesium⅐nucleotide diphosphate-dependent stimulation of NDSUR1 is abnormal.…”

“…Like other members of the largest family of eukaryotic membrane transport proteins (4), SUR possess two non-equivalent nucleotide-binding domains, NBD1 and NBD2. Magnesium⅐nucleotide-bound NBD1/NBD2 dimers counterbalance a magnesium-independent nucleotide inhibition of the K ATP pore, an effect essentially saturated in intact cells by ATP present at Ͼ100 times the IC 50(ATP) (5). Consistent with this mechanism, loss-of-stimulation mutations in NBD of SUR1 (ABCC8), the neuroendocrine-type receptor, have been discovered in infants with persistent hyperinsulinemic hypoglycemia (6), whereas mutations in ABCC8 that overactivate K IR 6.2 (KCNJ11) in millimolar MgATP have been shown to cause permanent or transient neonatal diabetes, including ND with neurological symptoms (7).…”

Pore loop-mutated rat KIR6.1 and KIR6.2 suppress KATPcurrent in rat cardiomyocytes

“…Assembly of KIR6.1/KIR6.2 with SUR1 also lowers the single channel conductance (6). Native channels from human atrial and neonatal rat ventricular myocytes display a calculated channel conductance between 49 and 63 pS (5). A relatively low single channel conductance has also been measured in Purkinje cells (20) and rabbit ventricular myocytes (14), raising the possibility of KIR6.1/KIR6.2 heteromultimers in different regions of the heart.…”