Characterization of a novel Ca2+-Activated potassium channel in rat brain rough endoplasmic reticulum.

OBJECTIVES: Potassium channels in the endoplasmic reticulum (ER) are crucial for maintaining calcium balance during calcium fluxes. Disruption in ER calcium balance leads to ER stress, implicated in diseases like diabetes and Alzheimer's disease (AD). However, limited data exists on ER potassium channels in excitable tissues such as the brain. To fill this gap, we aimed to evaluate potassium currents in rat brain rough endoplasmic reticulum (RER). METHODS: Rats were euthanized under deep anesthesia and their brains were immediately removed. The brains were then homogenized in ice-cold sucrose buffer, followed by the extraction of RER microsomes through a series of centrifugation processes. Purity of sample was evaluated using western blotting technique. Single channel recordings were done in voltage steps from +50 to -60 mV following incorporation of rat brain RER vesicles into planar bilayers. RESULTS: We observed a voltage-dependent potassium channel with an approximate conductance of 188 pS. Channel open probability was low at negative voltages, increasing at positive voltages. The channel was blocked by Charybdotoxin but not by Iberiotoxin. Additionally, TRAM-34, a specific KCa3.1 channel blocker, suppressed channel current amplitude and open probability. Western blot analysis revealed specific bands for anti-KCa3.1 antibody, approximately 50 kDa in brain homogenate and RER fraction. CONCLUSION: Our study provides strong evidence for the presence of an KCa3.1 channel on the RER membrane in rat brain, exhibiting distinct electro-pharmacological profile compared to plasma membrane and other organelles.

Association of Serum Vitamin D Levels with the Risk of Suicidal Behavior in Ilam, Iran: A Brief Report.

There are conflicting reports on the effect of serum vitamin D (VD) levels on the development of suicidal behavior. VD deficiency is prevalent in Ilam province, and this region has the highest suicide mortality rate in Iran. The present study aimed to evaluate a possible association between serum VD levels and the risk of suicide among the inhabitants of Ilam province. A total of 157 suicide attempters (case group) and 314 age- and sex-matched individuals (control group) without a history of suicide attempts were recruited into the study. Suicide attempters were admitted to the Emergency Department of Ilam Shahid Mostafa Khomeini Hospital (Ilam, Iran) between March 2018 and March 2019. The individuals in the control group were randomly selected from those referred to various medical laboratories in Ilam during the same period. The participants in both groups were aged 18-35 years, and none had a history of kidney, liver, or endocrine diseases. Serum VD levels were measured using the enzyme-linked immunosorbent assay method. Categorical and continuous variables were compared using the Chi square test and independent samples t test, respectively. Serum VD level in the control group (31.5±0.2 ng/mL) was significantly lower than in the case group (43.1±0.1 ng/mL) (P<0.001). The results of logistic regression analysis showed that an increase in serum VD score was associated with an increase in the likelihood of suicide attempts (OR=1.05, 95% CI=1.03-1.06, P<0.001). Our findings confirmed the role of VD deficiency in the development of suicidal behavior. However, it is not a significant factor in its pathogenesis.

GFAP and Neuron Specific Enolase (NSE) in the Serum of Suicide Attempters.

Background: To determine whether neuronal damage and/or neuroinflammation exist in the brain of suicide attempters and to find a novel biological biomarker to help distinguishing high risk individuals with suicide behavior, we aimed to measure glial fibrillary acidic protein (GFAP), neuron specific enolase (NSE), and nerve growth factor (NGF) in suicide attempters. Methods: In the present case-control study, the serum level of NSE, GFAP, and NGF were measured quantitatively in 43 suicide attempters and 43 healthy control participants aged 18 to 35 years. Data were analyzed using the nonpaired t test followed by the Mann-Whitney posttest. Results: The mean serum level of NSE and GFAP were significantly higher in suicide attempters compared with healthy control individuals (p = 0.003, p = 0.001, respectively), while no significant difference was detected in NGF serum level between the 2 groups. Conclusion: Our findings of increased level of NSE along with the significant increase in GFAP would propose the presence of low grade neuroinflammation in the brain of these participants. NSE/GFAP might be good markers that is easily accessible and can be considered as prognostic markers in high-risk suicide attempters.

The Role of Oxidant-Antioxidant Status in Suicide Behavior in Kurdish Ethnicity.

INTRODUCTION: Oxidative stress plays a critical role in the pathogenesis of neurodegenerative and neuropsychiatric disorders. However, its role in suicidal behavior has not been clarified yet. Consequently, we aimed to evaluate the oxidant-antioxidant status in the serum of suicide attempters in Ilam city. METHODS: Fifty suicide attempters and 40 control subjects (volunteers) aged 18-35 years were studied in the current experiment. To consider the oxidant-antioxidant status, serum levels of Malondialdehyde (MDA), Nitric Oxide (NO), Superoxide Dismutase (SOD), and the Total Antioxidant Capacity (TAC) were measured. RESULTS: Serum levels of SOD and TAC were significantly lower in the suicide attempters group compared to the controls. Furthermore, serum NO level was significantly higher in the suicide attempters compared to the control groups. Interestingly, the serum level of MDA was significantly lower in the suicide attempters compared to the control groups. CONCLUSION: The oxidative stress without MDA elevation, detected in suicide attempters, can be considered a biochemical hallmark in suicide behavior.

Evidence for a KATP Channel in Rough Endoplasmic Reticulum (rerKATP Channel) of Rat Hepatocytes.

We report in a previous study the presence of a large conductance K+ channel in the membrane of rough endoplasmic reticulum (RER) from rat hepatocytes incorporated into lipid bilayers. Channel activity in this case was found to decrease in presence of ATP 100 µM on the cytoplasmic side and was totally inhibited at ATP concentrations greater than 0.25 mM. Although such features would be compatible with the presence of a KATP channel in the RER, recent data obtained from a brain mitochondrial inner membrane preparation have provided evidence for a Maxi-K channel which could also be blocked by ATP within the mM concentration range. A series of channel incorporation experiments was thus undertaken to determine if the ATP-sensitive channel originally observed in the RER corresponds to KATP channel. Our results indicate that the gating and permeation properties of this channel are unaffected by the addition of 800 nM charybdotoxin and 1 µM iberiotoxin, but appeared sensitive to 10 mM TEA and 2.5 mM ATP. Furthermore, adding 100 µM glibenclamide at positive potentials and 400 µM tolbutamide at negative or positive voltages caused a strong inhibition of channel activity. Finally Western blot analyses provided evidence for Kir6.2, SUR1 and/or SUR2B, and SUR2A expression in our RER fractions. It was concluded on the basis of these observations that the channel previously characterized in RER membranes corresponds to KATP, suggesting that opening of this channel may enhance Ca2+ releases, alter the dynamics of the Ca2+ transient and prevent accumulation of Ca2+ in the ER during Ca2+ overload.

Gating behavior of endoplasmic reticulum potassium channels of rat hepatocytes in diabetes.

BACKGROUND: Defects in endoplasmic reticulum homeostasis are common occurrences in different diseases, such as diabetes, in which the function of endoplasmic reticulum is disrupted. It is now well established that ion channels of endoplasmic reticulum membrane have a critical role in endoplasmic reticulum luminal homeostasis. Our previous studies showed the presence of an ATP-sensitive cationic channel in endoplasmic reticulum. Therefore, in this study, we examined and compared the activities of this channel in control and diabetic rats using single-channel recording techniques. METHOD: Male Wistar rats were made diabetic for 2 weeks with a single dose injection of streptozotocin (45 mg/kg). Ion channel incorporation of rough endoplasmic reticulum of diabetic hepatocytes into the bilayer lipid membrane allowed the characterization of K+ channel. RESULTS: Ion channel incorporation of rough endoplasmic reticulum vesicles into the bilayer lipid revealed that the channel current-voltage (I-V) relation with a mean slope conductance of 520 ± 19 pS was unaffected in diabetes. Interestingly, the channel Po-voltage relation was significantly lower in diabetic rats at voltages above +30 mV. CONCLUSION: We concluded that the endoplasmic reticulum cationic channel is involved in diabetes. Also, this finding could be considered as a goal for further therapeutic plans.

Exploration of human, rat, and rabbit embryonic cardiomyocytes suggests K-channel block as a common teratogenic mechanism.

AIMS: Several drugs blocking the rapidly activating potassium (K(r)) channel cause malformations (including cardiac defects) and embryonic death in animal teratology studies. In humans, these drugs have an established risk for acquired long-QT syndrome and arrhythmia. Recently, associations between cardiac defects and spontaneous abortions have been reported for drugs widely used in pregnancy (e.g. antidepressants), with long-QT syndrome risk. To investigate whether a common embryonic adverse-effect mechanism exists in the human, rat, and rabbit embryos, we made a comparative study of embryonic cardiomyocytes from all three species. METHODS AND RESULTS: Patch-clamp and quantitative-mRNA measurements of K(r) and slowly activating K (K(s)) channels were performed on human, rat, and rabbit primary cardiomyocytes and cardiac samples from different embryo-foetal stages. The K(r) channel was present when the heart started to beat in all species, but was, in contrast to human and rabbit, lost in rats in late organogenesis. The specific K(r)-channel blocker E-4031 prolonged the action potential in a species- and development-dependent fashion, consistent with the observed K(r)-channel expression pattern and reported sensitive periods of developmental toxicity. E-4031 also increased the QT interval and induced 2:1 atrio-ventricular block in multi-electrode array electrographic recordings of rat embryos. The K(s) channel was expressed in human and rat throughout the embryo-foetal period but not in rabbit. CONCLUSION: This first comparison of mRNA expression, potassium currents, and action-potential characteristics, with and without a specific K(r)-channel blocker in human, rat, and rabbit embryos provides evidence of K(r)-channel inhibition as a common mechanism for embryonic malformations and death.