Temporary Sequestration of Potassium by Mitochondria in Astrocytes

Michael G Kozoriz,John Church,Mark A Ozog,Christian C Naus,Claudia Krebs

doi:10.1074/jbc.m109.082073

Michael G Kozoriz, John Church + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.082073

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Abstract

Increases in extracellular potassium concentration ([K(+)](o)), which can occur during neuronal activity and under pathological conditions such as ischemia, lead to a variety of potentially detrimental effects on neuronal function. Although astrocytes are known to contribute to the clearance of excess K(+)(o), the mechanisms are not fully understood. We examined the potential role of mitochondria in sequestering K(+) in astrocytes. Astrocytes were loaded with the fluorescent K(+) indicator PBFI and release of K(+) from mitochondria into the cytoplasm was examined after uncoupling the mitochondrial membrane potential with carbonyl cyanide m-chlorophenylhydrazone (CCCP). Under the experimental conditions employed, transient applications of elevated [K(+)](o) led to increases in K(+) within mitochondria, as assessed by increases in the magnitudes of cytoplasmic [K(+)] ([K(+)](i)) transients evoked by brief exposures to CCCP. When mitochondrial K(+) sequestration was impaired by prolonged application of CCCP, there was a robust increase in [K(+)](i) upon exposure to elevated [K(+)](o). Blockade of plasmalemmal K(+) uptake routes by ouabain, Ba(2+), or a mixture of voltage-activated K(+) channel inhibitors reduced K(+) uptake into mitochondria. Also, reductions in mitochondrial K(+) uptake occurred in the presence of mito-K(ATP) channel inhibitors. Rises in [K(+)](i) evoked by brief applications of CCCP following exposure to high [K(+)](o) were also reduced by gap junction blockers and in astrocytes isolated from connexin43-null mice, suggesting that connexins also play a role in K(+) uptake into astrocyte mitochondria. We conclude that mitochondria play a key role in K(+)(o) handling by astrocytes.

Highlights

MM under resting conditions, [Kϩ]o can increase to Ͼ10 mM during seizure activity, Ͼ25 mM during ischemia, and Ͼ50 mM during spreading depression, with consequent effects on neuronal excitability, synaptic transmission, neurotransmitter reuptake and, neuronal viability
The entry of Kϩ across the mitochondrial inner membrane into mitochondria occurs via a Kϩ leak pathway dependent on the ⌬⌿m and a Kϩ uniporter, mito-KATP, whereas excess Kϩ can be removed by a Kϩ/Hϩ antiporter [16]; calcium-sensitive mitochondrial potassium currents have been described [33]. To determine whether these mechanisms contribute to Kϩo uptake into astrocyte mitochondria, the effects of a variety of inhibitors were examined on the changes in [Kϩ]i evoked by 5-min exposures to 12.5 mM Kϩo during prolonged mitochondrial uncoupling with carbonyl cyanide m-chlorophenylhydrazone (CCCP), where mitochondrial Kϩ sequestration is impaired, and on the [Kϩ]i transient induced by a 2-min application of CCCP immediately after a 5-min exposure to 12.5 mM Kϩo
During prolonged exposures to CCCP, CBX and AGA failed to inhibit the rise in [Kϩ]i normally evoked by exposure to 12.5 mM Kϩo (Fig. 7, A and B) suggesting, in turn, that connexon hemichannels do not play a role in Kϩ uptake at the level of the plasma membrane

Summary

Introduction

MM under resting conditions, [Kϩ]o can increase to Ͼ10 mM during seizure activity, Ͼ25 mM during ischemia, and Ͼ50 mM during spreading depression, with consequent effects on neuronal excitability, synaptic transmission, neurotransmitter reuptake and, neuronal viability (reviewed in Refs. 1 and 2). Because mitochondrial [Kϩ] was decreasing in response to the majority of our experimental conditions, in situ calibration experiments (see Ref. 28) were performed by exposing astrocytes to either 1.5 or 65 mM [Naϩ]i (the latter approximating the rise in [Naϩ]i following a 5-min exposure to CCCP; see supplemental Fig. S2).

Results

Conclusion