Nucleotide-gated K_ATPchannels integrated with creatine and adenylate kinases: Amplification, tuning and sensing of energetic signals in the compartmentalized cellular environment

Abstract

Transmission of energetic signals to membrane sensors, such as the ATP-sensitive K + (K ATP ) channel, is vital for cellular adaptation to stress. Yet, cell compartmentation implies diffusional hindrances that hamper direct reception of cytosolic energetic signals. With high intracellular ATP levels, K ATP channels may sense not bulk cytosolic, but rather local submembrane nucleotide concentrations set by membrane ATPases and phosphotransfer enzymes. Here, we analyzed the role of adenylate kinase and creatine kinase phosphotransfer reactions in energetic signal transmission over the strong diffusional barrier in the submembrane compartment, and translation of such signals into a nucleotide response detectable by K ATP channels. Facilitated diffusion provided by creatine kinase and adenylate kinase phosphotransfer dissipated nucleotide gradients imposed by membrane ATPases, and shunted diffusional restrictions. Energetic signals, simulated as deviation of bulk ATP from its basal level, were amplified into an augmented nucleotide response in the submembrane space due to failure under stress of creatine kinase to facilitate nucleotide diffusion. Tuning of creatine kinase-dependent amplification of the nucleotide response was provided by adenylate kinase capable of adjusting the ATP/ADP ratio in the submembrane compartment securing adequate K ATP channel response in accord with cellular metabolic demand. Thus, complementation between creatine kinase and adenylate kinase systems, here predicted by modeling and further supported experimentally, provides a mechanistic basis for metabolic sensor function governed by alterations in intracellular phosphotransfer fluxes. KeywordsATP-sensitive K + channel; nucleotide diffusion; metabolic sensor; intracellular compartment; heart Metabolic sensing by K ATP channelsMaintenance of homeostasis requires efficient transmission of energetic signals from sites of ATP generation to ATP sensors governing cellular response [1][2][3][4][5][6]. In the compartmentalized cell environment, energetic signaling must integrate detection, amplification and delivery of metabolic signals arising from deviations in adenine nucleotide levels [1][2][3][4][5][6][7][8][9]. While the identity of energy-responsive elements is being increasingly resolved, the molecular mechanisms that synchronize metabolic sensor function with cell metabolism remain largely unknown. © 2004 Kluwer Academic PublishersAddress for offprints: A.E. Alekseev, Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Guggenheim 7, Rochester, MN 55905, USA (E-mail: alekseev.alexey@mayo.edu). (Fig. 1A) [18][19][20]. The sensor role of cardiac K ATP channels stems from the nonequivalent properties of nucleotide binding domains (NBD1 and NBD2) in the SUR2A subunit (Fig. 1A). NBD1 binds nucleotides whereas NBD2 hydrolyzes ATP, with NBDs working in tandem to gate K ATP channels [21][22][23]. The ATP hydrolysis cycle at SUR2A drives conformational transitio...

ATP-sensitive K channel channel/enzyme multimer: Metabolic gating in the heart

“…Indeed, the rate of ATP hydrolysis at NBD2 can be doubled by creatine kinase, reversing MgADP-induced K ATP channel opening highlighting a critical role for phosphotransfer reactions in setting the balance between the lifetime/probability of MgADP versus MgATP-bound conformational intermediates in the SUR ATPase cycle [9,14]. As a consequence of metabolic stress, a drop of creatine kinase flux compromises ADP removal from ATPase sites and suspends the ATPase cycle in a MgADP-bound state producing channel opening [12,15,16].…”

“…Indeed, during a normally operating ATPase, NBD2 during each single cycle unavoidably adopts the ADPbound conformation that potentially could activate K ATP channels. Paradoxically, ATPregenerating systems that facilitate ATPase cycling and therefore promote transit through the MgADP-bound intermediate state, do not activate K ATP channels [9,15,16,45]. It is only through arrest of the SUR ATPase in a pre-or post-hydrolytic conformation that specific channel outcome, pore closure or antagonism of ATP-induced pore inhibition, can be achieved [9].…”

“…Theoretical and experimental analysis of cooperative interaction between the two prototypic phosphotransfer enzymes, creatine kinase (CK) and adenylate kinase (AK) systems, in a compartmentalized cellular environment reveals unique properties of these enzymes in transmission of energetic signals to sarcolemmal K ATP channels [8,15,16]. Specifically, signal transmission from the cytosol to the submembrane compartment is limited due to restricted diffusion of nucleotides, creatine (Cr) and creatine phosphate (CrP).…”

“…Under cardiomyocyte stress, phosphotransfer flux through CK is significantly damped [92][93][94] such that with only a moderate drop in bulk cytosolic ATP, CK could no longer effectively dissipate nucleotide gradients precipitating a significant fall in submembrane CrP, and generation of an amplified nucleotide response at the K ATP channel site (Fig. 2) [16]. According to the CK reaction equilibrium, substitution by creatine of 9.95 out of the total 10.25 mM of creatine phosphate, a 50 μM drop of bulk ATP (from 0.3 to 0.25 mM) and concomitant increase of bulk ADP can be expected.…”

“…The basic gating of the K ATP channel that underlies the channel's metabolic response occurs in reaction to the balance at the channel site of inhibitory and stimulatory nucleotides, ATP and ADP, respectively [1,14]. The way in which the cellular metabolic state is "read" incorporates generation and delivery of nucleotide signals to the K ATP channel subunits, and nucleotide interactions with specialized channel domains that ultimately secure signal processing and translation into pore gating [15,16].…”