Exploring the molecular mechanisms of AMPK-dependent regulation of neuroexcitability

The AMP-activated protein kinase (AMPK) is an energy sensor that maintains cellular metabolic homeostasis in a cell-autonomous manner during energy stress conditions, such as hypoxia and glucose deprivation. Emerging evidence suggests that AMPK activation during metabolic stress conditions may have the capacity to directly phosphorylate and regulate the activity of non-metabolic targets outside its canonical pathways controlling metabolic homeostasis, such as receptors, and ion channels, thus modulating cellular functions in a cell-specific manner. This thesis aimed to examine the effect of AMPK activation on two distinct ion channels: human TWIK-related acid-sensitive potassium (hTASK-3) channels and murine hyperpolarization-activated cyclic nucleotide-gated (mHCN) channels. hTASK-3 channels belong to the family of two pore domain potassium (K2P) channels, which are voltage- and time-independent channels that are constitutively open over the entire range of physiological membrane potentials. Heteromeric TASK-1/3 channels are critical to oxygen sensing in carotid body type 1 cells, where hypoxia-induced inhibition of TASK-3 and/or TASK-1/3 potassium currents leads to voltage-gated calcium entry, exocytotic transmitter release and increases in carotid body afferent input responses that initiate corrective changes in breathing patterns. It was proposed that, in response to hypoxia, AMPK might directly phosphorylate and inhibit TASK channels, in particular TASK-3, but studies on rat type I cells questioned this view. However, sequence alignment identified a putative AMPK recognition motif in hTASK-3, but not hTASK-1, with Ser55 representing a potential phosphorylation site. I therefore studied the effects of five different AMPK activators on recombinant hTASK-3 potassium channels expressed in HEK-293 cells. Two structurally unrelated AMPK activators, the thienopyridine A-769662 (>100 µM) and the benzimidazole 991 (>3 µM), significantly inhibited hTASK-3 currents in a concentration-dependent manner, while the 4-azabenzimidazole MK-8722 (>3 µM) partially inhibited hTASK-3 at concentrations above those required for maximal AMPK activation. By contrast, the 4-azabenzimidazole BI-9774 (10-100 µM; a closely related analogue of MK-8722) and the pro-drug AICA-riboside (1 mM; metabolised to ZMP, an AMP-mimetic) had no significant effect on hTASK-3 currents at concentrations sufficient to maximally activate AMPK. Importantly, A-769662 (300 µM) also inhibited hTASK-3 channel currents in HEK-293 cells that stably overexpress an AMPK β-1 subunit mutant (S108A) that renders AMPK insensitive to activators that bind to the Allosteric Drug and Metabolite (ADaM) site, such as A-769662. These findings provide evidence that A-769662 and 991 may act as novel hTASK-3 channel inhibitors and suggest that AMPK does not regulate hTASK-3 channel currents. Unlike voltage-gated potassium channels, HCN channels are activated by hyperpolarization, and they are non-selective potassium channels, conducting a mixed inward current of Na+ and K+ ions. They are widely expressed in excitable cells, where they play vital functions in controlling resting membrane potential, spontaneous firing, synaptic integration and neurotransmitter release. Given their high energy demands, I sought to determine whether AMPK has the capacity to modulate the activity of different HCN isoforms (HCN1-4) to conserve energy status. Using whole-cell voltage clamp and two different experimental approaches, including acute application and/or preincubation, I studied the effect of different AMPK activators, such as MK-8722, 991, and/or compound 13 on the biophysical properties of mHCN1-4 channels stably expressed in HEK-293 cells. I showed that AMPK activation significantly induced a hyperpolarizing shift in the half-maximal activation voltage (G0.5) and slowed the activation kinetics of the first evolved HCN isoform, the mHCN3 channel. The pan AMPK activator MK-8722 also significantly accelerated mHCN3 channel deactivation. Importantly, all these effects were reduced by the pan AMPK inhibitor BAY-3827. Interestingly, my data also demonstrated that AMPK activation opposed the effect of cyclic adenosine monophosphate (cAMP) on the voltage-dependent gating, as well as the activation and deactivation kinetics of mHCN2 channels. Specifically, AMPK activation significantly induced a hyperpolarizing shift in G0.5 values, slowed down the activation kinetics. MK-8722 also significantly accelerated cAMP-modulated mHCN2 channel deactivation kinetics. By contrast, no effect was observed on the biophysical properties of mHCN1 or mHCN4 channels. These findings suggest that AMPK activation can differentially regulate the activity of HCN channel subtypes. In conclusion, the current study provides the first evidence that AMPK activation has the capacity to differentially regulate members of the HCN channel family. While AMPK activation significantly altered the gating and kinetics of mHCN2 channels (but only in the presence of cAMP) and mHCN3 channels, it showed no effect on hTASK-3, mHCN1 and mHCN4 channels. These findings offer valuable insights into the regulatory mechanisms of AMPK of the ion channel activity and underscore its potential role in maintaining energy homeostasis by regulating neuroexcitability.

Grantor dc:publisher

The University of Edinburgh

Year dc:date.issued

2025

Author dc:creator

• Said, Esraa

Advisors dc:contributor.advisor

• Evans, Mark
• Theil, Thomas
• Livingstone, Dawn

• TASK channels
• HCN channels
• AMPK

Language dc:language.iso

en

Identifier URI

http://dx.doi.org/10.7488/era/6501

OAI identifier oai:identifier

oai:era.ed.ac.uk:1842/43971