RESUMO
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.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Canais de Potássio Shab/metabolismo , Potenciais de Ação , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ativação Enzimática , Humanos , Ionomicina/farmacologia , Fosforilação , RatosRESUMO
A novel L-glutamate-gated anion channel (IscaGluCl1) has been cloned from the black-legged tick, Ixodes scapularis, which transmits multiple pathogens including the agents of Lyme disease and human granulocytic anaplasmosis. When mRNA encoding IscaGluCl1 was expressed in Xenopus laevis oocytes, we detected robust 50-400â¯nA currents in response to 100⯵M L-glutamate. Responses to L-glutamate were concentration-dependent (pEC50 3.64⯱â¯0.11). Ibotenate was a partial agonist on IscaGluCl1. We detected no response to 100⯵M aspartate, quisqualate, kainate, AMPA or NMDA. Ivermectin at 1⯵M activated IscaGluCl1, whereas picrotoxinin (pIC50 6.20⯱â¯0.04) and the phenylpyrazole fipronil (pIC50 6.90⯱â¯0.04) showed concentration-dependent block of the L-glutamate response. The indole alkaloid okaramine B, isolated from fermentation products of Penicillium simplicissimum (strain AK40) grown on okara pulp, activated IscaGluCl1 in a concentration-dependent manner (pEC50 5.43⯱â¯0.43) and may serve as a candidate lead compound for the development of new acaricides.