Phosphorylation of the voltage-gated potassium channel Kv2.1 by AMP-activated protein kinase regulates membrane excitability

Abstract

Firing of action potentials in excitable cells accelerates ATP turnover. The voltage-gated potassium channel Kv2.1 regulates action potential frequency in central neurons, whereas the ubiquitous cellular energy sensor AMP-activated protein kinase (AMPK) is activated by ATP depletion and protects cells by switching off energy-consuming processes. We show that treatment of HEK293 cells expressing Kv2.1 with the AMPK activator A-769662 caused hyperpolarizing shifts in the current-voltage relationship for channel activation and inactivation. We identified two sites (S440 and S537) directly phosphorylated on Kv2.1 by AMPK and, using phosphospecific antibodies and quantitative mass spectrometry, show that phosphorylation of both sites increased in A-769662-treated cells. Effects of A-769662 were abolished in cells expressing Kv2.1 with S440A but not with S537A substitutions, suggesting that phosphorylation of S440 was responsible for these effects. Identical shifts in voltage gating were observed after introducing into cells, via the patch pipette, recombinant AMPK rendered active but phosphatase-resistant by thiophosphorylation. Ionomycin caused changes in Kv2.1 gating very similar to those caused by A-769662 but acted via a different mechanism involving Kv2.1 dephosphorylation. In cultured rat hippocampal neurons, A-769662 caused hyperpolarizing shifts in voltage gating similar to those in HEK293 cells, effects that were abolished by intracellular dialysis with Kv2.1 antibodies. When active thiophosphorylated AMPK was introduced into cultured neurons via the patch pipette, a progressive, time-dependent decrease in the frequency of evoked action potentials was observed. Our results suggest that activation of AMPK in neurons during conditions of metabolic stress exerts a protective role by reducing neuronal excitability and thus conserving energy.calcineurin | calcium signaling | energy homeostasis A MP-activated protein kinase (AMPK) is a ubiquitously expressed sensor of cellular energy status (1). It is activated in response to increases in cellular AMP:ATP and ADP:ATP ratios by a mechanism involving allosteric activation and increased net phosphorylation at a conserved threonine (Thr172) mediated by the tumor-suppressor kinase, LKB1 (2). Thr172 phosphorylation and activation also can be triggered by increases in cytoplasmic Ca 2+ via the calmodulin-dependent kinase calcium/calmodulin kinase kinase β (CaMKKβ) (1, 2). Although AMPK initially was thought to maintain cellular energy homeostasis primarily by regulating metabolism, emerging evidence suggests that it also modulates cell function by phosphorylating other targets, including ion channels. This function may be of particular significance in excitable cells such as central neurons. Remarkably, ATP turnover in rodent brain is comparable with that in human leg muscle during marathon running, and it has been estimated that action potentials account for 25-50% of this turnover, with synaptic transmission (triggered by action potentials) accounting for all but 15% o...

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