Hypoxic Depolarization of Cerebellar Granule Neurons by Specific Inhibition of TASK-1

Abstract

Background and Purpose-The mechanisms underlying neuronal excitotoxicity during hypoxic/ischemic episodes are not fully understood. One feature of such insults is a rapid and transient depolarization of central neurons. TASK-1, an open rectifying K ϩ leak channel, is significant in setting the resting membrane potential of rat cerebellar granule neurons by mediating a standing outward K ϩ current. In this study we investigate the theory that the transient neuronal depolarization seen during hypoxia is due to the inhibition of TASK-1. Methods-Activity of TASK-1 in primary cultures of rat cerebellar granule neurons was investigated by the whole-cell patch-clamp technique. Discriminating pharmacological and electrophysiological maneuvers were used to isolate the specific channel types underlying acute hypoxic depolarizations. Results-Exposure of cells to acute hypoxia resulted in a reversible and highly reproducible mean membrane depolarization of 14.2Ϯ2.6 mV (nϭ5; PϽ0.01). Two recognized means of inhibiting TASK-1 (decreasing extracellular pH to 6.4 or exposure to the TASK-1-selective inhibitor anandamide) abolished both the hypoxic depolarization and the hypoxic depression of a standing outward current, identifying TASK-1 as the channel mediating this effect. Conclusions-Our data provide compelling evidence that hypoxia depolarizes central neurons by specific inhibition of TASK-1. Since this hypoxic depolarization may be an early, contributory factor in the response of central neurons to hypoxic/ischemic episodes, TASK-1 may provide a potential therapeutic target in the treatment of stroke.

Sumoylation of Na V 1.2 Channels Mediates the Early Response to Acute Hypoxia in Central Neurons

“…Thus, increased I Na may be self-limiting (or protective) if it leads to membrane depolarization, inducing Na V channel inactivation and, thereby, limits subsequent Na + flux. Consistent with this notion, we previously found that acute hypoxic challenge induces membrane depolarization when rat CGN were studied in current-clamp mode (Plant et al, 2002). Indeed, chronic stressors that increase the level of SUMOylated proteins in cells (Shao et al, 2004), including the transcription factor HIF1a (Cheng et al, 2007), and modify global patterns of SUMOylation (Cimarosti et al, 2012) have been inferred to be cytoprotective in cases where gene regulation and protein turnover are altered to conserve energy (Henley et al, 2014).…”

“…Because parallel fibers are rich in Na V 1.2 channels, our studies may underestimate the effects of Na V 1.2-SUMOylation in response to hypoxia in the cerebellum. Moreover, we employ standard, potassium-free, recording solutions to study I Na and, therefore, I Kso , a rat CGN current we previously showed to be responsive to hypoxia (Plant et al, 2002), was not evaluated in this work.…”

“…DOI: 10.7554/eLife.20054.004 À67 ± 2 mV, the last parameter a measure of the number of channels available to pass current (Table 1). When O 2 was lowered from ambient levels to 5% by perfusion of cells with hypoxic solutions (Plant et al, 2002), the mean peak I Na increased over 40 s to a new, stable level that was~70% higher, À294 ± 25 pA/pF ( Figure 1a and Table 1), reminiscent of increases in I Na in response to acute hypoxia reported by others studying rat neurons from the hypothalamus (Horn and Waldrop, 2000) and hippocampus (Raley-Susman et al, 2001). Augmentation of I Na by hypoxia was associated with a leftward shift of À11 ± 2 mV in both V ½ and SSI, allowing the same amount of depolarization to evoke larger I Na currents (Figure 1b).…”

“…The rat b1 accessory subunit was co-expressed with Na V 1.2 subunits unless otherwise indicated. CGN were cultured from 6 to 8 day old Sprague-Dawley rat pups (Charles River, Wilmington, MA; RRID:RGD_734476) as previously described (Plant et al, 2002). Briefly, the cerebellum was isolated, cut into~300 mm cubes, triturated with a fire-polished Pasteur pipette and incubated for 15 min at 37˚C with 2.5 mg/ml trypsin in PBS.…”

Widespread depolarization during expiration: A source of respiratory drive?

“…These studies suggest that lack of oxygen may lead to depolarization and reduced levels of oxygen may lead to partial depolarization. Studies have shown that hypoxia leads to depolarization in different types of rat cells [73,74], as well as depolarization of mitochondria [75,76]. This hypoxic depolarization of mitochondria appears to be caused by decreased electron transport chain activity due to lack of oxygen [76].…”