KCNQ1, KCNE2, and Na+-coupled solute transporters form reciprocally regulating complexes that affect neuronal excitability.

Na(+)-coupled solute transport is crucial for the uptake of nutrients and metabolic precursors, such as myo-inositol, an important osmolyte and precursor for various cell signaling molecules. We found that various solute transporters and potassium channel subunits formed complexes and reciprocally regulated each other in vitro and in vivo. Global metabolite profiling revealed that mice lacking KCNE2, a K(+) channel ß subunit, showed a reduction in myo-inositol concentration in cerebrospinal fluid (CSF) but not in serum. Increased behavioral responsiveness to stress and seizure susceptibility in Kcne2(-/-) mice were alleviated by injections of myo-inositol. Suspecting a defect in myo-inositol transport, we found that KCNE2 and KCNQ1, a voltage-gated potassium channel α subunit, colocalized and coimmunoprecipitated with SMIT1, a Na(+)-coupled myo-inositol transporter, in the choroid plexus epithelium. Heterologous coexpression demonstrated that myo-inositol transport by SMIT1 was augmented by coexpression of KCNQ1 but was inhibited by coexpression of both KCNQ1 and KCNE2, which form a constitutively active, heteromeric K(+) channel. SMIT1 and the related transporter SMIT2 were also inhibited by a constitutively active mutant form of KCNQ1. The activities of KCNQ1 and KCNQ1-KCNE2 were augmented by SMIT1 and the glucose transporter SGLT1 but were suppressed by SMIT2. Channel-transporter signaling complexes may be a widespread mechanism to facilitate solute transport and electrochemical crosstalk.

Amiloride but not memantine reduces neurodegeneration, seizures and myoclonic jerks in rats with cardiac arrest-induced global cerebral hypoxia and reperfusion.

It has been reported that both activation of N-methyl-D-aspartate receptors and acid-sensing ion channels during cerebral ischemic insult contributed to brain injury. But which of these two molecular targets plays a more pivotal role in hypoxia-induced brain injury during ischemia is not known. In this study, the neuroprotective effects of an acid-sensing cation channel blocker and an N-methyl-D-aspartate receptor blocker were evaluated in a rat model of cardiac arrest-induced cerebral hypoxia. We found that intracisternal injection of amiloride, an acid-sensing ion channel blocker, dose-dependently reduced cerebral hypoxia-induced neurodegeneration, seizures, and audiogenic myoclonic jerks. In contrast, intracisternal injection of memantine, a selective uncompetitive N-methyl-D-aspartate receptor blocker, had no significant effect on cerebral hypoxia-induced neurodegeneration, seizure and audiogenic myoclonic jerks. Intracisternal injection of zoniporide, a specific sodium-hydrogen exchanger inhibitor, before cardiac arrest-induced cerebral hypoxia, also did not reduce cerebral hypoxia-induced neurodegeneration, seizures and myoclonic jerks. These results suggest that acid-sensing ion channels play a more pivotal role than N-methyl-D-aspartate receptors in mediating cerebral hypoxia-induced brain injury during ischemic insult.

Idebenone induces apoptotic cell death in the human dopaminergic neuroblastoma SHSY-5Y cells.

Idebenone is a coenzyme Q10 analog and an antioxidant that has been used clinically to treat Friedreich Ataxia. Being an antioxidant, idebenone could have potential therapeutic potential to treat other neurodegenerative diseases such as Parkinson's disease in which oxidative stress plays a role in their pathogenesis. But whether idebenone can be used to treat Parkinson's disease has not been evaluated. In this study, we found that exposure of the dopaminergic neuroblastoma SHSY-5Y cells to 1-10 µM idebenone for 72 h had no effect on the cell viability revealed by trypan blue exclusion assay and MTT assay. However, cells exposed to 25 µM or higher concentrations of idebenone showed extensive trypan blue-positive staining and significant reduction in cell viability revealed by MTT assay indicating that most of the cells were no longer viable. Idebenone-induced cell death was characterized by genomic DNA fragmentation and accumulation of cytochrome c in the cytosol indicating that the death was apoptotic in nature. In addition, idebenone induced an increase in the total RNA of the pro-apoptosis protein BAX, it also increased the caspase-3 activity in the cell lysates when compared with the untreated control cells or cells exposed to 10 µM or lower concentrations of idebenone. The detrimental effect of idebenone was attenuated by glutathione, an antioxidant, suggesting that oxidative stress contributed to the idebenone-induced cell death. In conclusion, our results suggest that antioxidant idebenone induced apoptosis when used in high concentrations.

(-)-Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, reduces dichlorodiphenyl-trichloroethane (DDT)-induced cell death in dopaminergic SHSY-5Y cells.

Results from epidemiological studies indicated that there exists an inverse correlation between consumption of green tea and neurodegenerative diseases including Parkinson's disease. We hypothesized that consumption of green tea would activate endogenous protective mechanisms against environmental toxin-induced cell injury, which is believed to play a causative role in the etiology of Parkinson's disease. Here, we found that epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, concentration-dependently (1 microM, 3 microM and 10 microM) reduced dichlorodiphenyl-trichloroethane (DDT) (100 microM)-induced cell death in dopaminergic neuroblastoma SHSY-5Y cells. The cell viability was determined by trypan blue exclusion assays. We also found that preconditioning the SHSY-5Y cells with EGCG by multiple, brief, prior exposures of the cells to EGCG can subsequently protect the cells from DDT-induced cell death. The EGCG-induced protective effect positively correlated with the number of exposures to EGCG. These results suggest that EGCG has a protective effect against DDT-induced cell death, and that prior exposures to EGCG activate an endogenous protective mechanism in the dopaminergic cells which can mitigate organochlorine pesticide-induced cell injury.

Memantine exacerbates myoclonic jerks in a rat model of posthypoxic myoclonus.

The mechanism of cerebral hypoxia-induced myoclonic jerks is not known. Some studies have suggested that glutaminergic NMDA receptor activation in the inferior olive resulting in excitotoxic neuronal injury in the cerebellum is the underlying cause of posthypoxic myoclonus. To test this hypothesis, the effect of memantine, an NMDA receptor antagonist, on the intensity of myoclonic jerks and the extent of cerebral ischemia-induced neurodegeneration in the cerebellum were evaluated in a rat model of posthypoxic myoclonus. The myoclonus scores for the posthypoxic rats treated with memantine were significantly higher than those treated with saline. The myoclonic scores for the posthypoxic rats injected with 100mg/kg memantine are higher than those posthypoxic rats injected with 30 mg/kg memantine. In contrast, the number of Fluoro-Jade B positive degenerating neurons in the Purkinje cell layer of the cerebellum did not differ significantly between the memantine-treated and the saline-treated posthypoxic rats. This pattern of results suggests that glutaminergic NMDA receptor activation in the cerebellum does not play a significant role in the generation of myoclonus in a rat model of posthypoxic myoclonus. Further, these results also suggest that NMDA receptor antagonists would exacerbate posthypoxic myoclonus in this animal model.

Voltage-dependent C-type inactivation in a constitutively open K+ channel.

Most voltage-gated potassium (Kv) channels undergo C-type inactivation during sustained depolarization. The voltage dependence and other mechanistic aspects of this process are debated, and difficult to elucidate because of concomitant voltage-dependent activation. Here, we demonstrate that MinK-KCNQ1 (I(Ks)) channels with an S6-domain mutation, F340W in KCNQ1, exhibit constitutive activation but voltage-dependent C-type inactivation. F340W-I(Ks) inactivation was sensitive to extracellular cation concentration and species, and it altered ion selectivity, suggestive of pore constriction. The rate and extent of F340W-I(Ks) inactivation and recovery from inactivation were voltage-dependent with physiologic intracellular ion concentrations, and in the absence or presence of external K(+), with an estimated gating charge, z(i), of approximately 1. Finally, double-mutant channels with a single S4 charge neutralization (R231A,F340W-I(Ks)) exhibited constitutive C-type inactivation. The results suggest that F340W-I(Ks) channels exhibit voltage-dependent C-type inactivation involving S4, without the necessity for voltage-dependent opening, allosteric coupling to voltage-dependent S6 transitions occurring during channel opening, or voltage-dependent changes in ion occupancy. The data also identify F340 as a critical hub for KCNQ1 gating processes and their modulation by MinK, and present a unique system for further mechanistic studies of the role of coupling of C-type inactivation to S4 movement, without contamination from voltage-dependent activation.

Ketogenic diet prevents seizure and reduces myoclonic jerks in rats with cardiac arrest-induced cerebral hypoxia.

Although the mechanism underlying the anti-epileptic effects of a ketogenic diet (KD) is not known, KD is reported to be an effective treatment for intractable epilepsy, in particular among children. Here, we evaluated whether a KD can reduce posthypoxic seizure and myoclonic jerks in a rat model of cardiac arrest-induced cerebral hypoxia. In this study, rats were divided into two groups: one group received a normal diet while the other group was fed a KD for 25 days before being subjected to cardiac arrest-induced cerebral hypoxia. We found that rats fed a normal diet developed seizures and severe myoclonic jerks in response to auditory stimuli after the hypoxic insults, whereas the rats on the KD did not develop seizure and showed much less severe myoclonic jerks in response to auditory stimuli. The results suggested that the KD has beneficial effects against posthypoxic seizure and myoclonus.

Post-hypoxic animal model of myoclonus.

Post-hypoxic myoclonus is a form of myoclonus frequently caused by cardiac arrest. Development of an animal model may facilitate understanding of the condition and its treatments. We describe an animal model of post-hypoxic myoclonus developed in our laboratory through cardiac arrest, initially induced by chemical and later by mechanical obstruction of major cardiac vessels. These animals respond to valproate, clonazepam and 5-hydroxytrytophan reminiscent of its human counterpart. We review their behavioral, pharmacological and neuropathological features. Therapy developed for myoclonus in this model may be helpful for myoclonus from other etiologies such as corticobasal degeneration, Lewy-body disorders, Creutzfeld-Jacob disease, Alzheimer's disease.

NMDA receptor-mediated excitotoxicity contributes to the cerebral hypoxic injury of a rat model of posthypoxic myoclonus.

Cardiac arrest-induced cerebral hypoxic injury could induce posthypoxic movement disorders. Here we investigated the effects of memantine, an NMDA receptor channel blocker, on the neurodegeneration occurred in an established rat model of posthypoxic myoclonus. We found that administration of memantine for 7 days significantly reduced cerebral hypoxia-induced neurodegeneration in the CA1 of the hippocampus, the reticular thalamic nucleus (RTN) and the primary fissure of the cerebellum of the posthypoxic animals. The results suggest that the neurodegeneration observed in specific areas of the brain of the posthypoxic rats is contributed by NMDA receptor-mediated excitotoxicity.

Interaction of KCNE subunits with the KCNQ1 K+ channel pore.

KCNQ1 alpha subunits form functionally distinct potassium channels by coassembling with KCNE ancillary subunits MinK and MiRP2. MinK-KCNQ1 channels generate the slowly activating, voltage-dependent cardiac IKs current. MiRP2-KCNQ1 channels form a constitutively active current in the colon. The structural basis for these contrasting channel properties, and the mechanisms of alpha subunit modulation by KCNE subunits, are not fully understood. Here, scanning mutagenesis located a tryptophan-tolerant region at positions 338-340 within the KCNQ1 pore-lining S6 domain, suggesting an exposed region possibly amenable to interaction with transmembrane ancillary subunits. This hypothesis was tested using concomitant mutagenesis in KCNQ1 and in the membrane-localized 'activation triplet' regions of MinK and MiRP2 to identify pairs of residues that interact to control KCNQ1 activation. Three pairs of mutations exerted dramatic effects, ablating channel function or either removing or restoring control of KCNQ1 activation. The results place KCNE subunits close to the KCNQ1 pore, indicating interaction of MiRP2-72 with KCNQ1-338; and MinK-59,58 with KCNQ1-339, 340. These data are consistent either with perturbation of the S6 domain by MinK or MiRP2, dissimilar positioning of MinK and MiRP2 within the channel complex, or both. Further, the results suggest specifically that two of the interactions, MiRP2-72/KCNQ1-338 and MinK-58/KCNQ1-340, are required for the contrasting gating effects of MinK and MiRP2.

Post-hypoxic myoclonus induces Fos expression in the reticular thalamic nucleus and neurons in the brainstem.

Post-hypoxic myoclonus is a movement disorder characterized by brief, sudden involuntary muscle jerks. Although the mechanism underlying this disorder remains unclear, earlier pharmacological studies indicated that aberrant activity of specific neuronal circuitry in the central nervous system causes this disorder. In the present study, Fos protein, an immediate-early gene product, was used as a marker of neuronal activity to identify the brain nuclei possibly involved in post-hypoxic myoclonus. We found that Fos protein was immunologically detected in the reticular thalamic nucleus (RT), the medial longitudinal fasciculus (MLF) as well as in the locus coeruleus (LC) and the periventricular gray substance (PVG) in post-hypoxic rats that developed myoclonus in response to auditory stimuli. Fos was not detected in these nuclei from rats that underwent 4 min of cardiac arrest without myoclonus. Electrolytic lesions of the RT or MLF but not the LC/PVG significantly reduced auditory stimulated myoclonus in the post-hypoxic rats. The results suggest that neuronal activity in the RT and the MLF plays a contributing role in post-hypoxic myoclonus.

Activation of mitochondrial ATP-sensitive potassium channels increases cell viability against rotenone-induced cell death.

We recently showed that activation of ATP-sensitive potassium (KATP) channels in PC12 cells induces protection against the neurotoxic effect of rotenone, a mitochondrial complex I inhibitor. In this study, we sought to determine the locus of the KATP channels that mediate this protection in PC12 cells. We found that pretreatment of PC12 cells with diazoxide, a mitochondrial KATP channel selective opener, dose-dependently increases cell viability against rotenone-induced cell death as indicated in trypan blue exclusion assays. The protective effect of this preconditioning is attenuated by 5-hydroxydecanoic acid (5-HD), a selective mitochondrial KATP channel antagonist but not in the presence of HMR-1098, a selective plasma membrane KATP potassium channel antagonist. In contrast, P-1075, a selective plasma membrane KATP channel opener, does not induce protection. Using specific antibodies against SUR1 and Kir6.1, we detected immunoreactive proteins of apparent molecular masses 155 and 50 kDa, corresponding to those previously reported for SUR1 and Kir6.1, respectively, in the mitochondria-enriched fraction of PC12 cells. In addition, whole cell patch-clamp studies revealed that inward currents in PC12 cells are insensitive to P-1075, HMR-1098, glibenclamide and diazoxide, indicating that functional plasma membrane KATP channels are negligible. Taken together, our results demonstrate for the first time that activation of mitochondrial KATP channels elicits protection against rotenone-induced cell death.

Activation of adenosine triphosphate-sensitive potassium channels confers protection against rotenone-induced cell death: therapeutic implications for Parkinson's disease.

It is anticipated that further understanding of the protective mechanism induced by ischemic preconditioning will improve prognosis for patients of ischemic injury. It is not known whether preconditioning exerts beneficial actions in neurodegenerative diseases, in which ischemic injury plays a causative role. Here we show that transient activation of ATP-sensitive potassium channels, a trigger in ischemic preconditioning signaling, confers protection in PC12 cells and SH-SY5Y cells against neurotoxic effect of rotenone and MPTP, mitochondrial complex I inhibitors that have been implicated in the pathogenesis of Parkinson's disease. The degree of protection is in proportion to the bouts of exposure to an ATP-sensitive potassium channel opener, a feature reminiscent of ischemic tolerance in vivo. Protection is sensitive to a protein synthesis inhibitor, indicating the involvement of de novo protein synthesis in the protective processes. Pretreatment of PC12 cells with preconditioning stimuli FeSO(4) or xanthine/xanthine oxidase also confers protection against rotenone-induced cell death. Our results demonstrate for the first time the protective role of ATP-sensitive potassium channels in a dopaminergic neuronal cell line against rotenone-induced neurotoxicity and conceptually support the view that ischemic preconditioning-derived therapeutic strategies may have potential and feasibility in therapy for Parkinson's disease.