Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels

Abstract

Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K+ (K(ATP)) channels. However, the mechanism that communicates metabolic signals and integrates cellular energetics with K(ATP) channel-dependent membrane excitability remains elusive. Here, we identify that the response of K(ATP) channels to metabolic challenge is regulated by adenylate kinase phosphotransfer. Adenylate kinase associates with the K(ATP) channel complex, anchoring cellular phosphotransfer networks and facilitating delivery of mitochondrial signals to the membrane environment. Deletion of the adenylate kinase gene compromised nucleotide exchange at the channel site and impeded communication between mitochondria and K(ATP) channels, rendering cellular metabolic sensing defective. Assigning a signal processing role to adenylate kinase identifies a phosphorelay mechanism essential for efficient coupling of cellular energetics with K(ATP) channels and associated functions.

Figure 1

AMP-dependent regulation of K_ATP channels is lost in AK1-KO cardiac cells. (A) Recording of native cardiac K_ATP channels demonstrate activation of ATP-inhibited channels following application of AMP, a pivotal AK substrate. (B) Absence of the AMP effect in cardiomyocytes from AK1-KO mice. (C) Concentration dependence of AMP-induced activation on 250 μM ATP-inhibited K_ATP channels in control (○) and AK1-KO (●) cardiomyocytes.

Figure 2

CK regulates AK-mediated K_ATP channel activation. Although AMP activated both native cardiac (A) and recombinant Kir6.2/SUR2A (B) K_ATP channels, CrP, which promotes ADP scavenging by CK, antagonized AMP-induced K_ATP channel activation. (C) Average K_ATP channel activity (mean ± SE) in cardiomyocytes (n = 4; □) and in COS-7 cells expressing recombinant K_ATP channels (Kir6.2/SUR2A, n = 4; ▨), in the presence of 250 μM ATP, following addition of 50 μM AMP or following addition of AMP plus 2 mM CrP.

Figure 3

AK associates with sarcolemmal K_ATP channels. (A) Vigorous AK ATP ↔ ADP exchange activity in sarcolemma from wild-type (WT, n = 4; ▨), but not AK1-KO (n = 4; ■) mice. (B) Association of K_ATP channel protein with AK1 in wild-type, but not AK1-KO. Sarcolemmal K_ATP channels were immunoprecipitated (IP) with the anti-Kir6.2 Ab (Kir6.2Ab) and probed for AK1 by Western blot (WB) with the anti-AK1 Ab (AK1Ab). (C) HPLC chromatogram shows increased AK activity in K_ATP channel immunoprecipitates (IP) compared with control (C). The anti-Kir6.2 Ab was used to immunoprecipitate the channel complex from cardiac sarcolemma, while preimmune serum served as control. (D) Anti-AK1 Ab recognized, by Western blot, recombinant AK1 (left lane) and a protein of identical molecular mass in sarcolemmal Kir6.2 immunoprecipitates (center lane), which was competed off by excess of purified AK1 (right lane). (E) Channel proteins were in vitro translated and labeled with [³⁵S]methionine. In the presence of nonlabeled recombinant AK1, the anti-AK1 Ab immunoprecipitated Kir6.2 (left lane) or Kir6.2/SUR subunits (center lane). Channel proteins were not immunoprecipitated by a control Ab (right lane). Proteins were run on denaturing SDS/PAGE, and visualized by Western blotting (B and D) or autoradiography (E).

Figure 4

AK phosphotransfer under metabolic stress. (A) ³¹P NMR spectra of guinea pig heart extracts under normoxic and hypoxic conditions. Incorporation of ¹⁸O into β-phosphoryls of ADP, which reflects the rate of AK-catalyzed phosphotransfer, induces the appearance of three ¹⁸O-labeled species (¹⁸O₁, ¹⁸O₂, and ¹⁸O₃). Incorporation of ¹⁸O was significantly higher under hypoxia. (B) Average values for AK-catalyzed phosphotransfer flux in normoxia (n = 5; ▩) and hypoxia (n = 5; ■) in guinea pig heart determined by ¹⁸O/³¹P NMR spectroscopy. (C) AMP levels in wild-type (WT, n = 5; □) and AK1-KO (n = 5; ▨) mouse heart under normoxia and hypoxia determined by HPLC. (D) Blunted AK phosphotransfer response in the AK1-KO (n = 5; ░⃞) compared with the wild-type (n = 5; ■) mouse heart in hypoxia.

Figure 5

AK communicates mitochondrial signals to K_ATP channels. (A) The mitochondrial uncoupler, DNP, induced vigorous K_ATP channel activation which was irreversibly inhibited by oligomycin, a mitochondrial F_oF₁-ATPase antagonist (n = 5). (B) In the presence of CrP, DNP produced lower activation of K_ATP channels as part of mitochondria-generated ADP was scavenged by the CrP/CK system. Ap5A, which at 20 μM inhibited AK activity in cardiac sarcolemma (n = 3; C), suppressed AK-mediated channel activation (n = 5). (D) In AK1-KO cardiomyocytes, K_ATP channel activation by DNP was blunted and insensitive to Ap5A, but inhibited by oligomycin (n = 3). (E) Average (mean ± SE) DNP-induced K_ATP channel activity in wild-type (left) and AK1-KO (right) cardiomyocytes in 0.5 mM ATP (□) or 0.5 mM ATP plus 1 mM CrP (▨). Bars were constructed from three to five recordings for each data point.

Figure 6

AK phosphotransfer communicates mitochondria-generated signals to K_ATP channels. AK molecules form a phosphorelay network connecting mitochondria with the cell membrane. Under hypoxic stress, mitochondrial F_oF₁-ATPase consumes cellular ATP generating ADP, which is delivered to the K_ATP channel through the chain of sequential AK-catalyzed phosphotransfer reactions. The inwardly rectifying potassium channel (Kir6.2) and the SUR are the pore-forming and regulatory subunits of the K_ATP channel complex.