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1 Department of Physiology, Akita University School of Medicine, and
2 Core Research for Evolutional Science and Technology, Japan Science and Technology Cooperation, 1-1-1 Hondo, Akita 010-8543, Japan
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Abstract |
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Introduction |
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During seizure, the cerebral metabolic rates of O2 and glucose uptake increase more than under any other circumstance (2). This massive energy demand causes a rapid fall in ATP that, if prolonged, leads ultimately to irreversible cell damage (5) due to intracellular ionic derangements such as Na+ and Ca2+ overload (2). To prevent the development in the brain of energy-demanding seizure during metabolic stress, the ATP-sensitive K+ (KATP) channel, the molecule that controls membrane potentials by sensing intracellular ATP levels, may play a pivotal role. In this brief review, recent progress in accord with this hypothesis is discussed, together with other views.
KATP channels, discovered in cardiac myocytes and then found in many other excitable cells, including hormone-secreting cells, skeletal and smooth muscle cells, and neurons, alter open probability as the cytosolic ATP concentration changes (for review, see Ref. 14). The function of KATP channels has been described best in insulin-secreting pancreatic ß-cells (Fig. 1
). Elevated blood glucose increases the intracellular ATP/ADP ratio in ß-cells to close the channels, depolarizing the plasma membrane and activating the voltage-dependent Ca2+ channels, allowing Ca2+ influx to induce exocytosis of insulin. The sulfonylureas used in the treatment of diabetes mellitus also close the KATP channels to stimulate insulin secretion. In heart cells, on the other hand, the decreased cytosolic ATP concentration during ischemia or hypoxia promotes K+ efflux from the cells by activating the KATP channels, which rapidly dampens excitability by shortening the action potential duration.
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A key question remains from these extensive studies: what is the physiological role of brain KATP channels? Recent investigations of KATP channels by molecular approaches provide many insights. The structure of the KATP channel was initially determined in pancreatic ß-cells to be an octameric complex of two types of subunit (7): a pore-forming channel subunit (Kir6.2) and a regulatory subunit, the sulfonylurea receptor (SUR1), belonging to the ATP-binding cassette superfamily. Later, additional Kir and SUR subunits were identified that form complexes with different pharmacological properties: the cardiac and skeletal muscle types are composed of Kir6.2 and SUR2A, an isoform of SUR1 (8); the vascular smooth muscle type is composed of Kir6.1 and SUR2B, a splice variant of SUR2A (for review, see Ref. 14). SUR2A and SUR2B have low affinity for sulfonylurea, whereas SUR1 (ß-cell type) has high affinity.
The role of brain KATP channels during hypoxic challenge was investigated by using mutant mice lacking Kir6.2 [Kir6.2(–/–) mice] (18). The midbrain nucleus, called the substantia nigra pars reticulata (SNr), which consists mostly of GABAergic neurons, was selected as a focus partly because the nucleus expresses the highest binding densities for sulfonylureas with high affinity, suggesting that the KATP channels in SNr neurons are ß-cell-type KATP channels (Kir6.2/SUR1). Expression of ß-cell-type KATP channels in GABAergic SNr neurons has been verified functionally by single-channel analyses (see Ref. 18 and references therein). In addition, Liss and coworkers (10) have demonstrated by single-cell RT-PCR that the SNr GABAergic neurons solely express pancreatic ß-cell-type KATP channels. Thus the function of SNr GABAergic neurons in Kir6.2(–/–) mice should yield clues to the roles of ß-cell-type KATP channels in the brain. Another reason is that the SNr is thought to act as a central gate in the propagation of generalized seizure (3). Pharmacological inhibition or selective bilateral lesions of the SNr suppress seizure spread in most animal models of epilepsy. In addition, anticonvulsant drugs that enhance the GABA-mediated inhibition of seizures and the blockade of excitatory neurotransmission in the nucleus raise the threshold for seizures (for review, see Ref. 3). The possible involvement of the KATP channels of the SNr in seizure control was first mentioned by Amoroso and colleagues in 1990 (1). They suggested that a decrease in blood glucose suppresses GABA release from the nerve terminals in the substantia nigra by hyperpolarization due to the opening of presynaptic KATP channels and proposed that this decrease in the inhibitory capacity of the GABA system during hypoglycemia might affect seizure protection by the SN. Thus altered seizure susceptibility might be expected in Kir6.2(–/–) mice.
Daily behavior and basal physiological parameters of Kir6.2(–/–) mice in resting conditions were not significantly different from those of wild-type mice. However, during brief hypoxia (150 s, 5.4% O2), Kir6.2(–/–) mice all exhibited a myoclonic jerk in <10 s followed by severe tonic-chronic convulsion and death at 22 s on average, whereas wild-type mice all remained sedated during the challenge and revived normally. Electroencephalogram (EEG) and electromyogram (EMG) revealed a sequence of seizure patterns in conscious knockout mice: very-low-voltage EEG traces for a few seconds indicating loss of consciousness, then fast waves for several seconds after an abrupt, sharp deflection corresponding to the tonic convulsion and myoclonus, followed by bilateral, high-voltage sharp wave bursts. In wild-type mice under the same conditions, a medium-to-low-voltage EEG trace predominated during the hypoxic challenge, suggesting that the KATP channels participate critically in protection from seizure. This is supported by a recent study using SUR1-overexpressing animals (6). Under the control of a Ca2+-calmodulin kinase promoter, these transgenic mice overexpress the SUR1 subunit in forebrain. These mice show a significant increase in the threshold for kainate-induced seizures together with increased survival rate, suggesting that KATP channels play a pivotal role in raising the threshold for seizures caused not only by metabolic deficiency but also by excitotoxicity.
How do KATP channels control the seizure threshold? To investigate the cellular and ionic mechanisms, single unit activities were recorded in the SNr by acute slice preparations in Kir6.2(–/–) and wild-type mice (18). The spontaneous firing rate of the SNr neurons under resting conditions was similar in both mice. However, during brief hypoxic challenge, the wild-type neurons showed a marked decrease in the firing rate to about one-third, whereas the firing rate of knockout neurons increased ~1.8-fold. In addition, the sulfonylurea tolbutamide reversed the hypoxia-induced inhibition of the firing of wild-type neurons to facilitation just as in Kir6.2(–/–) neurons, although tolbutamide had no effect on the firing rate or the membrane potential of SNr neurons under resting conditions. During brief hypoxia, the membrane potentials of wild-type SNr neurons were shifted in the hyperpolarized direction, whereas Kir6.2(–/–) SNr neurons showed no hyperpolarization but rather were depolarized in nystatin perforated-patch recordings using dissociated SNr neurons (18). These results indicate that the opening of the KATP channels exerts a strong suppressive effect on wild-type SNr neuronal activity during hypoxic challenge by shifting membrane potentials in the hyperpolarized direction sufficiently to reverse the facilitation of neuronal activity in the Kir6.2(–/–) neurons that is due to membrane depolarization (Fig. 1
). The mechanism of the spike facilitation and membrane depolarization observed in Kir6.2(–/–) SNr neurons is currently unknown; depression of electrogenic Na+-K+ pump activity during hypoxic challenge is most likely, but other mechanisms such as inactivation of O2-sensitive K+ channels by decreased PO2 are possible (4).
As mentioned, presynaptic modulatory effects on neuronal firing by brain KATP channels have been suggested during metabolic stress (1,16,17). In the substantia nigra pars compacta (SNc), the KATP channels on the striatonigral terminals of SNc dopaminergic neurons might become active due to reduced intraterminal levels of ATP, and the resultant hyperpolarization of the terminal should lead to a reduction of GABA release and so facilitate SNc neuronal activity. However, as pointed out by Watts and colleagues (17), it is difficult to reconcile this hypothesis with the postsynaptic suppressive effects of KATP channel activation. The contribution of postsynaptic KATP channels in neuronal responses to hypoxic challenge was investigated in acute slice preparations (18). In the condition of isolation from presynaptic effects by the presence of blockers of both excitatory and inhibitory fast neurotransmissions, the firing rates of both wild-type and Kir6.2(–/–) GABAergic SNr neurons increased ~35% in normoxia, indicating some facilitatory effect of blocked presynaptic GABAergic terminals on the firings of SNr neurons. However, the responses to hypoxic challenge in the presence and absence of blockers were similar: a striking contrast in the spontaneous firing rate of SNr neurons and a net decrease in wild-type and a net increase in Kir6.2(–/–) neurons, indicating that the opening of the postsynaptic KATP channels is pivotal in the hypoxia-induced responses of wild-type SNr neurons, at least in this experimental condition. The physiological significance of the presynaptic KATP channels must be further investigated.
It is important to note that wild-type mice exhibited generalized convulsion in very severe hypoxic conditions, such as 4.3% O2 for 150 s (18). In milder hypoxic conditions, such as 7.3% O2, most of the Kir 6.2(–/–) mice showed no convulsion and none died. Secher and Wilhjelm (13) have reported that the tolerance of conscious animals to anoxia increases very rapidly when the O2 concentration is >5%, whereas at <4% O2 survival time is extremely short (<10 min). There is a rapid deflection in the O2 concentration vs. survival time curve at ~4–5% O2. Thus the KATP channel may fully exert its protective role only in limited severities of hypoxic challenge just above this critical transition. In addition, the EEG and EMG of Kir6.2(–/–) mice responded within several seconds after the hypoxic condition was achieved, suggesting involvement of the KATP channels in the initial stage of the response to hypoxic challenge.
To investigate the contribution of the KATP channels in other brain nuclei to the hypoxia-induced response is important. Although KATP channels are functionally expressed in various nuclei, such as cerebral cortex (11), hippocampus (19), hypothalamus, and SNc (reviewed in Ref. 10), the molecular makeup of neuronal KATP channels appears not to be homogeneous. Liss and colleagues (10), using a combined approach of patch-clamp and single-cell RT-PCR, reported that dopaminergic SNc neurons express different types of KATP channel with differing sensitivities to metabolic inhibition and proposed a novel mechanism of the selective vulnerability of some dopaminergic neurons in Parkinson's disease. They showed that neurons with ß-cell-type KATP channels, which comprise Kir6.2 and SUR1, have the highest metabolic sensitivity and that these and not neurons with other types of KATP channels survive in weaver mice, suggesting that the ß-cell-type KATP channels might have the strongest neuroprotective effect. Zawar and colleagues (20) also reported heterogeneous expression profiles of KATP channels in the hippocampal CA1 area: functional KATP channels (Kir6.1 plus SUR1, Kir6.2 plus SUR1 or SUR2) are expressed in 17% of the pyramidal cells and 75% of the interneurons. Especially interesting, 58% of CA1 interneurons express ß-cell-type KATP channels. Clarification of the involvement of these channels in energy-depleted conditions should provide clues to understanding why certain sets of pyramidal neurons are extremely vulnerable to ischemic stress but others are not.
What then is the specific role of the KATP channels in the SNr? It is widely known that the neurons of the SNr show the highest spontaneous activity (up to 100 Hz) in the brain, indicating a very high metabolic rate in these neurons in the normoxic condition. Indeed, SNr neurons are extremely sensitive to hypoxia (18). On the other hand, it has been reported that the potentials evoked by electrical stimulation in hippocampal (5) and cerebral cortical (18) neurons are not altered during brief hypoxia. Thus the KATP channels in SNr neurons are likely to act as the sensors in hypoxia, responding before the general self-defense reaction to hypoxic conditions in other neuron types (Fig. 1). In addition, SNr neurons innervate various distant nuclei in diverse motor-related functions, including the ventral thalamic nuclei, superior colliculus, and pedunculopontine nucleus in the brain stem. Abrupt silence in the SNr GABAergic projection neurons during an early phase of brain metabolic emergency might well exert the nigral protection mechanism by conveying a signal of massive disinhibition to all of these targets simultaneously, which should protect the whole brain from generalized seizure. In addition, these studies suggest that KATP channels may be a target of site-specific treatment of brain disorders associated with ATP insufficiency such as stroke and metabolic encephalopathies.
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This article has been cited by other articles:
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  D. R. Johnson, J. C. O'Connor, M. E. Hartman, R. I. Tapping, and G. G. Freund Acute Hypoxia Activates the Neuroimmune System, Which Diabetes Exacerbates J. Neurosci., January 31, 2007; 27(5): 1161 - 1166. [Abstract] [Full Text] [PDF]
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 R. Bright and D. Mochly-Rosen The Role of Protein Kinase C in Cerebral Ischemic and Reperfusion Injury Stroke, December 1, 2005; 36(12): 2781 - 2790. [Abstract] [Full Text] [PDF]
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