Exploring the Impact of BKCa Channel Function in Cellular Membranes on Cardiac Electrical Activity.

This review paper delves into the current body of evidence, offering a thorough analysis of the impact of large-conductance Ca2+-activated K+ (BKCa or BK) channels on the electrical dynamics of the heart. Alterations in the activity of BKCa channels, responsible for the generation of the overall magnitude of Ca2+-activated K+ current at the whole-cell level, occur through allosteric mechanisms. The collaborative interplay between membrane depolarization and heightened intracellular Ca2+ ion concentrations collectively contribute to the activation of BKCa channels. Although fully developed mammalian cardiac cells do not exhibit functional expression of these ion channels, evidence suggests their presence in cardiac fibroblasts that surround and potentially establish close connections with neighboring cardiac cells. When cardiac cells form close associations with fibroblasts, the high single-ion conductance of these channels, approximately ranging from 150 to 250 pS, can result in the random depolarization of the adjacent cardiac cell membranes. While cardiac fibroblasts are typically electrically non-excitable, their prevalence within heart tissue increases, particularly in the context of aging myocardial infarction or atrial fibrillation. This augmented presence of BKCa channels' conductance holds the potential to amplify the excitability of cardiac cell membranes through effective electrical coupling between fibroblasts and cardiomyocytes. In this scenario, this heightened excitability may contribute to the onset of cardiac arrhythmias. Moreover, it is worth noting that the substances influencing the activity of these BKCa channels might influence cardiac electrical activity as well. Taken together, the BKCa channel activity residing in cardiac fibroblasts may contribute to cardiac electrical function occurring in vivo.

Enhancing Human Cutaneous Wound Healing through Targeted Suppression of Large Conductance Ca2+-Activated K+ Channels.

The modulation of K+ channels plays a crucial role in cell migration and proliferation, but the effect of K+ channels on human cutaneous wound healing (CWH) remains underexplored. This study aimed to determine the necessity of modulating K+ channel activity and expression for human CWH. The use of 25 mM KCl as a K+ channel blocker markedly improved wound healing in vitro (in keratinocytes and fibroblasts) and in vivo (in rat and porcine models). K+ channel blockers, such as quinine and tetraethylammonium, aided in vitro wound healing, while Ba2+ was the exception and did not show similar effects. Single-channel recordings revealed that the Ba2+-insensitive large conductance Ca2+-activated K+ (BKCa) channel was predominantly present in human keratinocytes. NS1619, an opener of the BKCa channel, hindered wound healing processes like proliferation, migration, and filopodia formation. Conversely, charybdotoxin and iberiotoxin, which are BKCa channel blockers, dramatically enhanced these processes. The downregulation of BKCa also improved CWH, whereas its overexpression impeded these healing processes. These findings underscore the facilitative effect of BKCa channel suppression on CWH, proposing BKCa channels as potential molecular targets for enhancing human cutaneous wound healing.

Rufinamide, a Triazole-Derived Antiepileptic Drug, Stimulates Ca2+-Activated K+ Currents While Inhibiting Voltage-Gated Na+ Currents.

Rufinamide (RFM) is a clinically utilized antiepileptic drug that, as a triazole derivative, has a unique structure. The extent to which this drug affects membrane ionic currents remains incompletely understood. With the aid of patch clamp technology, we investigated the effects of RFM on the amplitude, gating, and hysteresis of ionic currents from pituitary GH3 lactotrophs. RFM increased the amplitude of Ca2+-activated K+ currents (IK(Ca)) in pituitary GH3 lactotrophs, and the increase was attenuated by the further addition of iberiotoxin or paxilline. The addition of RFM to the cytosolic surface of the detached patch of membrane resulted in the enhanced activity of large-conductance Ca2+-activated K+ channels (BKCa channels), and paxilline reversed this activity. RFM increased the strength of the hysteresis exhibited by the BKCa channels and induced by an inverted isosceles-triangular ramp pulse. The peak and late voltage-gated Na+ current (INa) evoked by rapid step depolarizations were differentially suppressed by RFM. The molecular docking approach suggested that RFM bound to the intracellular domain of KCa1.1 channels with amino acid residues, thereby functionally affecting BKCa channels' activity. This study is the first to present evidence that, in addition to inhibiting the INa, RFM effectively modifies the IK(Ca), which suggests that it has an impact on neuronal function and excitability.

Functional characterization of ion channels expressed in kidney organoids derived from human induced pluripotent stem cells.

Kidney organoids derived from human or rodent pluripotent stem cells have glomerular structures and differentiated/polarized nephron segments. Although there is an increasing understanding of the patterns of expression of transcripts and proteins within kidney organoids, there is a paucity of data regarding functional protein expression, in particular on transporters that mediate the vectorial transport of solutes. Using cells derived from kidney organoids, we examined the functional expression of key ion channels that are expressed in distal nephron segments: the large-conductance Ca2+-activated K+ (BKCa) channel, the renal outer medullary K+ (ROMK, Kir1.1) channel, and the epithelial Na+ channel (ENaC). RNA-sequencing analyses showed that genes encoding the pore-forming subunits of these transporters, and for BKCa channels, key accessory subunits, are expressed in kidney organoids. Expression and localization of selected ion channels was confirmed by immunofluorescence microscopy and immunoblot analysis. Electrophysiological analysis showed that BKCa and ROMK channels are expressed in different cell populations. These two cell populations also expressed other unidentified Ba2+-sensitive K+ channels. BKCa expression was confirmed at a single channel level, based on its high conductance and voltage dependence of activation. We also found a population of cells expressing amiloride-sensitive ENaC currents. In summary, our results show that human kidney organoids functionally produce key distal nephron K+ and Na+ channels.NEW & NOTEWORTHY Our results show that human kidney organoids express key K+ and Na+ channels that are expressed on the apical membranes of cells in the aldosterone-sensitive distal nephron, including the large-conductance Ca2+-activated K+ channel, renal outer medullary K+ channel, and epithelial Na+ channel.

Evidence for Dual Activation of IK(M) and IK(Ca) Caused by QO-58 (5-(2,6-Dichloro-5-fluoropyridin-3-yl)-3-phenyl-2-(trifluoromethyl)-1H-pyrazolol[1,5-a]pyrimidin-7-one).

QO-58 (5-(2,6-dichloro-5-fluoropyridin-3-yl)-3-phenyl-2-(trifluoromethyl)-1H-pyrazolol[1,5-a]pyrimidin-7-one) has been regarded to be an activator of KV7 channels with analgesic properties. However, whether and how the presence of this compound can result in any modifications of other types of membrane ion channels in native cells are not thoroughly investigated. In this study, we investigated its perturbations on M-type K+ current (IK(M)), Ca2+-activated K+ current (IK(Ca)), large-conductance Ca2+-activated K+ (BKCa) channels, and erg-mediated K+ current (IK(erg)) identified from pituitary tumor (GH3) cells. Addition of QO-58 can increase the amplitude of IK(M) and IK(Ca) in a concentration-dependent fashion, with effective EC50 of 3.1 and 4.2 µM, respectively. This compound could shift the activation curve of IK(M) toward a leftward direction with being void of changes in the gating charge. The strength in voltage-dependent hysteresis (Vhys) of IK(M) evoked by upright triangular ramp pulse (Vramp) was enhanced by adding QO-58. The probabilities of M-type K+ (KM) channels that will be open increased upon the exposure to QO-58, although no modification in single-channel conductance was seen. Furthermore, GH3-cell exposure to QO-58 effectively increased the amplitude of IK(Ca) as well as enhanced the activity of BKCa channels. Under inside-out configuration, QO-58, applied at the cytosolic leaflet of the channel, activated BKCa-channel activity, and its increase could be attenuated by further addition of verruculogen, but not by linopirdine (10 µM). The application of QO-58 could lead to a leftward shift in the activation curve of BKCa channels with neither change in the gating charge nor in single-channel conductance. Moreover, cell exposure of QO-58 (10 µM) resulted in a minor suppression of IK(erg) amplitude in response to membrane hyperpolarization. The docking results also revealed that there are possible interactions of the QO-58 molecule with the KCNQ or KCa1.1 channel. Overall, dual activation of IK(M) and IK(Ca) caused by the presence of QO-58 eventually may have high impacts on the functional activity (e.g., anti-nociceptive effect) residing in electrically excitable cells. Care must be exercised when interpreting data generated with QO-58 as it is not entirely KCNQ/KV7 selective.

N-Methyl-D-Aspartic Acid Receptor-Mediated Vasodilation Is Attenuated in the Retinas of Diabetic Rats.

PURPOSE: Activation of N-methyl-d-aspartic acid (NMDA) receptors enhances nitric oxide (NO) production in retinal neuronal cells, and in turn, NO released from neuronal cells induces glial cell-mediated dilation of retinal arterioles in rats. The purpose of this study was to examine how neuronal cell-dependent, glial cell-mediated vasodilation is impacted in diabetic rat retinas. METHODS: Diabetes was induced in 6-week-old male Wistar rats by combining streptozotocin injection and D-glucose feeding. Two weeks later, the dilator function of retinal arterioles was assessed. RESULTS: Compared with non-diabetic rats, the dilator responses of retinal arterioles induced by intravitreal injection of NMDA and NOR3, an NO donor, were reduced in diabetic rats. Following the blockade of large-conductance Ca2+-activated K+ (BKCa) channels with iberiotoxin, no significant difference in the retinal vasodilator response to NOR3 was observed between non-diabetic and diabetic rats. Intravitreal injection of 14,15-epoxyeicosatrienoic acid, a vasodilatory factor released from glial cells, dilated retinal arterioles, and the response was diminished by diabetes. CONCLUSION: These findings suggest that the impaired BKCa channel function in vascular cells is responsible for the diminished neuronal cell-dependent, glial cell-mediated dilation of retinal arterioles during the early stage of diabetes.

Effective Activation of BKCa Channels by QO-40 (5-(Chloromethyl)-3-(Naphthalen-1-yl)-2-(Trifluoromethyl)Pyrazolo [1,5-a]pyrimidin-7(4H)-one), Known to Be an Opener of KCNQ2/Q3 Channels.

QO-40 (5-(chloromethyl)-3-(naphthalene-1-yl)-2-(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-7(4H)-one) is a novel and selective activator of KCNQ2/KCNQ3 K+ channels. However, it remains largely unknown whether this compound can modify any other type of plasmalemmal ionic channel. The effects of QO-40 on ion channels in pituitary GH3 lactotrophs were investigated in this study. QO-40 stimulated Ca2+-activated K+ current (IK(Ca)) with an EC50 value of 2.3 µM in these cells. QO-40-stimulated IK(Ca) was attenuated by the further addition of GAL-021 or paxilline but not by linopirdine or TRAM-34. In inside-out mode, this compound added to the intracellular leaflet of the detached patches stimulated large-conductance Ca2+-activated K+ (BKCa) channels with no change in single-channel conductance; however, there was a decrease in the slow component of the mean closed time of BKCa channels. The KD value required for the QO-40-mediated decrease in the slow component at the mean closure time was 1.96 µM. This compound shifted the steady-state activation curve of BKCa channels to a less positive voltage and decreased the gating charge of the channel. The application of QO-40 also increased the hysteretic strength of BKCa channels elicited by a long-lasting isosceles-triangular ramp voltage. In HEK293T cells expressing α-hSlo, QO-40 stimulated BKCa channel activity. Overall, these findings demonstrate that QO-40 can interact directly with the BKCa channel to increase the amplitude of IK(Ca) in GH3 cells.

The Integrated Effects of Brivaracetam, a Selective Analog of Levetiracetam, on Ionic Currents and Neuronal Excitability.

Brivaracetam (BRV) is recognized as a novel third-generation antiepileptic drug approved for the treatment of epilepsy. Emerging evidence has demonstrated that it has potentially better efficacy and tolerability than its analog, Levetiracetam (LEV). This, however, cannot be explained by their common synaptic vesicle-binding mechanism. Whether BRV can affect different ionic currents and concert these effects to alter neuronal excitability remains unclear. With the aid of patch clamp technology, we found that BRV concentration dependently inhibited the depolarization-induced M-type K+ current (IK(M)), decreased the delayed-rectifier K+ current (IK(DR)), and decreased the hyperpolarization-activated cation current in GH3 neurons. However, it had a concentration-dependent inhibition on voltage-gated Na+ current (INa). Under an inside-out patch configuration, a bath application of BRV increased the open probability of large-conductance Ca2+-activated K+ channels. Furthermore, in mHippoE-14 hippocampal neurons, the whole-cell INa was effectively depressed by BRV. In simulated modeling of hippocampal neurons, BRV was observed to reduce the firing of the action potentials (APs) concurrently with decreases in the AP amplitude. In animal models, BRV ameliorated acute seizures in both OD-1 and lithium-pilocarpine epilepsy models. However, LEV had effects in the latter only. Collectively, our study demonstrated BRV's multiple ionic mechanism in electrically excitable cells and a potential concerted effect on neuronal excitability and hyperexcitability disorders.

High ability of zileuton ((±)-1-(1-benzo[b]thien-2-ylethyl)-1-hydroxyurea) to stimulate IK(Ca) but suppress IK(DR) and IK(M) independently of 5-lipoxygenase inhibition.

Zileuton (Zyflo®) is regarded to be an inhibitor of 5-lipoxygenase. Although its effect on Ca2+-activated K+ currents has been reported, its overall ionic effects on neurons are uncertain. In whole-cell current recordings, zileuton increased the amplitude of Ca2+-activated K+ currents with an EC50 of 3.2 µM in pituitary GH3 lactotrophs. Furthermore, zileuton decreased the amplitudes of both delayed-rectifier K+ current (IK(DR)) and M-type K+ current (IK(M)). Conversely, no modification of hyperpolarization-activated cation current (Ih) was demonstrated in its presence of zileuton, although the subsequent addition of cilobradine effectively suppressed the current. In inside-out current recordings, the addition of zileuton to the bath increased the probability of large-conductance Ca2+-activated K+ (BKCa) channels; however, the subsequent addition of GAL-021 effectively reversed the stimulation of channel activity. The kinetic analyses showed an evident shortening in the slow component of mean closed time of BKCa channels in the presence of zileuton, with minimal change in mean open time or that in the fast component of mean closed time. The elevation of BKCa channels caused by zileuton was also observed in hippocampal mHippoE-14 neurons, without any modification of single-channel amplitude. In conclusion, except for its suppression of 5-lipoxygenase, our results indicate that zileuton does not exclusively act on BKCa channels, and its inhibitory effects on IK(DR) and IK(M) may combine to exert strong influence on the functional activities of electrically excitable cells in vivo.

Single Molecule Fluorescence Imaging Reveals the Stoichiometry of BKγ1 Subunit in Living HEK293 Cell Expression System.

Large conductance Ca2+-activated K+ (BKCa) channels are ubiquitously expressed in plasma membrane of both excitable and non-excitable cells and possess significant physiological functions. A tetrameric complex of α subunit (BKα) forms a functional pore of BKCa channel. The properties of BKCa channel, such as voltage-dependence, Ca2+ sensitivity and pharmacological responses, are extensively modulated by co-expressing accessory ß subunits (BKß), which can associate with BKα in one to one manner. Although the functional significance of newly identified γ subunits (BKγ) has been revealed, the stoichiometry between BKα and BKγ1 remains unclear. In the present study, we utilized a single molecule fluorescence imaging with a total internal reflection fluorescence (TIRF) microscope to directly count the number of green fluorescent protein (GFP)-tagged BKγ1 (BKγ1-GFP) within a single BKCa channel complex in HEK293 cell expression system. BKγ1-GFP significantly enhanced the BK channel activity even when the intracellular Ca2+ concentration was kept lower, i.e., 10 nM, than the physiological resting level. BKγ1-GFP stably formed molecular complexes with BKα-mCherry in the plasma membrane. Counting of GFP bleaching steps revealed that a BKCa channel can contain up to four BKγ1 per channel at the maximum. These results suggest that BKγ1 forms a BKCa channel complex with BKα in a 1 : 1 stoichiometry in a human cell line.

Involvement of Gi protein-dependent BKCa channel activation in ß2-adrenoceptor-mediated dilation of retinal arterioles in rats.

Circulating catecholamines contribute to the regulation of retinal vascular tone. Our previous studies have demonstrated that the activation of large-conductance Ca2+-activated K+ (BKCa) channels is involved in the ß2-adrenoceptor-mediated dilation of retinal arterioles in rats. The present study aimed to examine the role of Gi protein in the ß2-adrenoceptor-mediated activation of BKCa channels in the retinal arterioles. Images of in vivo rat ocular fundi were captured, and the diameters of retinal arterioles were measured. Systemic blood pressure and heart rate were recorded continuously. Intravenous infusion of formoterol (0.01-0.3 µg/kg/min), a ß2-adrenoceptor agonist, increased the diameter of retinal arterioles but decreased mean arterial pressure in a dose-dependent manner. Intravitreal injection of iberiotoxin (20 pmol/eye), an inhibitor of BKCa channels, significantly attenuated the formoterol-induced dilation of retinal arterioles. Similar results were obtained when salbutamol (0.03-3 µg/kg/min), another ß2-adrenoceptor agonist, was used instead of formoterol. However, iberiotoxin had no significant effect on retinal vasodilator responses to intravenous infusion of denopamine (1-30 µg/kg/min; a ß1-adrenoceptor agonist), CL316243 (0.3-10 µg/kg/min; a ß3-adrenoceptor agonist), prostaglandin I2 (0.03-10 µg/kg/min; a prostanoid IP receptor agonist), and forskolin (1-10 µg/kg/min; an adenylyl cyclase activator). Intravitreal injection of pertussis toxin (66 ng/eye; a Gi protein inhibitor) significantly attenuated the dilation of retinal arterioles induced by formoterol but not by denopamine and CL316243. In the presence of pertussis toxin, iberiotoxin had no inhibitory effect on formoterol-induced dilation of retinal arterioles. These results suggest that stimulation of ß2-adrenoceptors dilates retinal arterioles through pertussis toxin-sensitive Gi protein-dependent activation of BKCa channels in rats in vivo.

14-3-3γ, a novel regulator of the large-conductance Ca2+-activated K+ channel.

14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca2+-activated K+ (BK) channels revealed a putative 14-3-3 binding site in the COOH-terminal region. Our previous data showed that 14-3-3γ is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3γ has a novel role in the regulation of BK channel activity and protein expression. We used electrophysiology, Western blot analysis, and coimmunoprecipitation to examine the effects of 14-3-3γ on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3γ with BK α-subunits (BKα) by coimmunoprecipitation. In human embryonic kidney-293 cells stably expressing BKα, overexpression of 14-3-3γ significantly decreased BK channel activity and channel open probability. 14-3-3γ inhibited both total and cell surface BKα protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3γ and myc-BK. Knockdown of 14-3-3γ by siRNA transfection markedly increased BKα expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3γ-mediated inhibition of BK protein expression. Similarly, pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3γ on BK protein expression. Furthermore, overexpression of 14-3-3γ significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BKα. Additionally, 3 days of dietary K+ challenge reduced 14-3-3γ expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K+ excretion. These data suggest that 14-3-3γ modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal pathway.

Knockout of AKAP150 improves impaired BK channel-mediated vascular dysfunction through the Akt/GSK3ß signalling pathway in diabetes mellitus.

Vascular dysfunction resulting from diabetes is an important factor in arteriosclerosis. Previous studies have shown that during hyperglycaemia and diabetes, AKAP150 promotes vascular tone enhancement by intensifying the remodelling of the BK channel. However, the interaction between AKAP150 and the BK channel remains open to discussion. In this study, we investigated the regulation of impaired BK channel-mediated vascular dysfunction in diabetes mellitus. Using AKAP150 null mice (AKAP150-/- ) and wild-type (WT) control mice (C57BL/6J), diabetes was induced by intraperitoneal injection of streptozotocin. We found that knockout of AKAP150 reversed vascular remodelling and fibrosis in mice with diabetes and in AKAP150-/- diabetic mice. Impaired Akt/GSK3ß signalling contributed to decreased BK-ß1 expression in aortas from diabetic mice, and the silencing of AKAP150 increased Akt phosphorylation and BK-ß1 expression in MOVAS cells treated with HG medium. The inhibition of Akt activity caused a decrease in BK-ß1 expression, and treatment with AKAP150 siRNA suppressed GSK3ß expression in the nuclei of MOVAS cells treated with HG. Knockout of AKAP150 reverses impaired BK channel-mediated vascular dysfunction through the Akt/GSK3ß signalling pathway in diabetes mellitus.

High Efficacy by GAL-021: A Known Intravenous Peripheral Chemoreceptor Modulator that Suppresses BKCa-Channel Activity and Inhibits IK(M) or Ih.

: GAL-021 has recently been developed as a novel breathing control modulator. However, modifications of ionic currents produced by this agent remain uncertain, although its efficacy in suppressing the activity of big-conductance Ca2+-activated K+ (BKCa) channels has been reported. In pituitary tumor (GH3) cells, we found that the presence of GAL-021 decreased the amplitude of macroscopic Ca2+-activated K+ current (IK(Ca)) in a concentration-dependent manner with an effective IC50 of 2.33 µM. GAL-021-mediated reduction of IK(Ca) was reversed by subsequent application of verteporfin or ionomycin; however, it was not by that of diazoxide. In inside-out current recordings, the addition of GAL-021 to the bath markedly decreased the open-state probability of BKCa channels. This agent also resulted in a rightward shift in voltage dependence of the activation curve of BKCa channels; however, neither the gating charge of the curve nor single-channel conductance of the channel was changed. There was an evident lengthening of the mean closed time of BKCa channels in the presence of GAL-021, with no change in mean open time. The GAL-021 addition also suppressed M-type K+ current with an effective IC50 of 3.75 µM; however, its presence did not alter the amplitude of erg-mediated K+ current, or mildly suppressed delayed-rectifier K+ current. GAL-021 at a concentration of 30 µM could also suppress hyperpolarization-activated cationic current. In HEK293T cells expressing α-hSlo, the addition of GAL-021 was also able to suppress the BKCa-channel open probabilities, and GAL-021-mediated suppression of BKCa-channel activity was attenuated by further addition of BMS-191011. Collectively, the GAL-021 effects presented herein do not exclusively act on BKCa channels and these modifications on ionic currents exert significant influence on the functional activities of electrically excitable cells occurring in vivo.

Characterization of Effectiveness in Concerted Ih Inhibition and IK(Ca) Stimulation by Pterostilbene (Trans-3,5-dimethoxy-4'-hydroxystilbene), a Stilbenoid.

Pterostilbene (PTER), a natural dimethylated analog of resveratrol, has been demonstrated to produce anti-neoplastic or neuroprotective actions. However, how and whether this compound can entail any perturbations on ionic currents in electrically excitable cells remains unknown. In whole-cell current recordings, addition of PTER decreased the amplitude of macroscopic Ih during long-lasting hyperpolarization in GH3 cells in a concentration-dependent manner, with an effective IC50 value of 0.84 µM. Its presence also shifted the activation curve of Ih along the voltage axis to a more hyperpolarized potential, by 11 mV. PTER at a concentration greater than 10 µM could also suppress l-type Ca2+ and transient outward K+ currents in GH3 cells. With the addition of PTER, IK(Ca) amplitude was increased, with an EC50 value of 2.23 µM. This increase in IK(Ca) amplitude was attenuated by further addition of verruculogen, but not by tolbutamide or TRAM-39. Neither atropine nor nicotine, in the continued presence of PTER, modified the PTER-stimulated IK(Ca). PTER (10 µM) slightly suppressed the amplitude of l-type Ca2+ current and transient outward K+ current. The presence of PTER (3 µM) was also effective at increasing the open-state probability of large-conductance Ca2+-activated K+ (BKCa) channels identified in hippocampal mHippoE-14 neurons; however, its inability to alter single-channel conductance was detected. Our study highlights evidence to show that PTER has the propensity to perturb ionic currents (e.g., Ih and IK(Ca)), thereby influencing the functional activities of neurons, and neuroendocrine or endocrine cells.

Construction of stable cell lines expressing large conductance Ca2+-activated K+ channel α-subunit and molecular mechanism of potassium excretion in this channel / 上海交通大学学报（医学版）

Objective: To construct stable cell lines expressing the large conductance Ca2+-activated K+ channel (MaxiK or BK) α-subunit and to explore the mechanism of potassium excretion via BKα channel. Methods:The BKα plasmid with Myc tag was constructed and transfected into HEK293 cell lines by lipofectamine 2000. The positive monoclonal cell lines were screened by G418, and the expression of BKα was detected by Western blotting and the location of BKα by immunofluorescence. The stable cell lines expressing BKα protein was cultured on slides to form a single cell layer, which was perfused with different potassium ion concentrations of 5 mmol/L and 100 mmol/L, and the single channel patch clamp recorded the ion flux of BKα. Wild type and mutants (G77R, G130R, C140R and R297C) of the inwardly rectifying potassium channel (Kir4.1) were transfected into HEK293 cells stably transfected with BKα, and then the membrane protein was extracted. The expression of BKα was detected by Western blotting. Results:Stable cell lines expressing BKα channel were selected from HEK293 cells after transfection and cellular immunofluorescence verified the expression of BKα channel and its expression on the cell membrane. The channel open frequency (Npo) of BKα increased rapidly when perfused with 100 mmol/L potassium. After being transfected with wild type or mutants of Kir4.1, the membrane expression of BKα in the stable cell lines showed significant difference among these groups (P<0.05). Conclusion:The HEK293 cell lines stably expressing BKα have been successfully constructed. BKα channel can be activated by high potassium solutions. The function of the BKα subunit can be related to Kir4.1 channel, which may be attributed to the depolarization of the cells transfected by Kir4.1 mutants.

Effect of extracellular potassium on the activity of distal renal tubule in mice / 上海交通大学学报（医学版）

Objective: To investigate the effect of extracellular potassium (K+) concentration on the activities of sodium chloride co-transporter (NCC) and large conductance Ca2+-activated K+ channel (BK) in distal renal tubule of mice. Methods: Six specific pathogen free (SPF) C57BL/6 mice aged 8 to 10 weeks were sacrificed,and the kidney slices were made with previously reported method. Then,these slices were incubated randomly in normal K+,high K+,BaCl2 and RbCl solutions,respectively. The abundance and phosphorylation level of NCC in kidney slices at different K+ concentrations and different time courses were detected by Western blotting. The overall and membrane expressions of BK in kidney slices were also detected after incubation with different K+ solutions for 2 h. Results: Compared with normal K+ solution,NCC phosphorylation level was significantly decreased after incubation with high K+ solution for 5,15,30 min (all P<0.05),and NCC phosphorylation level was also decreased after intervention with K+ channel inhibitor Ba2+ or Rb+ (both P<0.05). After the treatment with high K+ solution for 2 h,neither the overall cell expression of BKα subunit and β4 subunit,nor membrane expression of BKα subunit was found significant changes compared with normal K+ incubation. Conclusion: High K+ can directly down-regulate NCC phosphorylation level,which may be preparation for kaliuresis of the downstream tubule of distal convoluted tubule.

Pioglitazone, a PPAR-γ Activator, Stimulates BKCa but Suppresses IK M in Hippocampal Neurons.

Pioglitazone (PIO), a thiazolidinedone, was reported to stimulate peroxisome proliferator-activated receptor-γ (PPAR-γ) with anti-inflammatory, anti-proliferative, anti-diabetic, and antidepressive activities. However, whether this compound exerts any perturbations on Ca2+-activated K+ and M-type K+ currents in central neurons remains largely unresolved. In this study, we investigated the effects of PIO on these potassium currents in hippocampal neurons (mHippoE-14). In whole-cell current recordings, the presence of PIO (10 µM) increased the amplitude of Ca2+-activated K+ current [IK(Ca)] in mHippoE-14 cells. PIO-induced stimulation of IK(Ca) observed in these cells was reversed by subsequent addition of paxilline, yet not by TRAM-39 or apamin. In inside-out current recordings, PIO applied to the bath concentration-dependently increased the activity of large-conductance Ca2+-activated K+ (BKCa) channels with an EC50 value of 7.6 µM. Its activation of BKCa channels in mHippoE-14 cells was voltage-dependent and accompanied by both a lengthening in mean open time and a shortening in slow component of mean closed time. The activation curve of BKCa channels after addition of PIO was shifted to less depolarized potential without any change in the gating charge. PIO also suppressed the amplitude of M-type K+ currents inherently in mHippoE-14 neurons. Taken together, in addition to its agonistic action on PPAR-γ, PIO-induced perturbation of these potassium channels may be responsible for its widely pharmacological actions on hippocampal neurons.

Dopamine D1 receptor-mediated upregulation of BKCa currents modifies Müller cell gliosis in a rat chronic ocular hypertension model.

Müller cell gliosis is a common response in many retinal pathological conditions. We previously demonstrated that downregulation of Kir channels contributes to Müller cell gliosis in a rat chronic ocular hypertension (COH) model. Here, the possible involvement of outward K+ currents in Müller cell gliosis was investigated. Outward K+ current densities in Müller cells isolated from COH rats, as compared with those in normal rats, showed a significant increase, which was mainly contributed by large-conductance Ca2+ -activated K+ (BKCa ) channels. The involvement of BKCa channels in Müller cell gliosis is suggested by the fact that glial fibrillary acidic protein (GFAP) levels were augmented in COH retinas when these channels were suppressed by intravitreal injections of iberiotoxin. In COH retinas an increase in dopamine (DA) D1 receptor (D1R) expression in Müller cells was revealed by both immunohistochemistry and Western blotting. Moreover, protein levels of tyrosine hydroxylase were also increased, and consistent to this, retinal DA contents were elevated. SKF81297, a selective D1R agonist, enhanced BKCa currents of normal Müller cells through intracellular cAMP-PKA signaling pathway. Furthermore, GFAP levels were increased by the D1R antagonist SCH23390 injected intravitreally through eliminating the BKCa current upregulation in COH retinas, but partially reduced by SKF81297. All these results strongly suggest that the DA-D1R system may be activated to a stronger extent in COH rat retinas, thus increasing BKCa currents of Müller cells. The upregulation of BKCa channels may antagonize the Kir channel inhibition-induced depolarization of Müller cells, thereby attenuating the gliosis of these cells.

Negative regulation of cellular Ca2+ mobilization by ryanodine receptor type 3 in mouse mesenteric artery smooth muscle.

Physiological functions of type 3 ryanodine receptors (RyR3) in smooth muscle (SM) tissues are not well understood, in spite of their wide expression. However, the short isoform of RyR3 is known to be a dominant-negative variant (DN-RyR3), which may negatively regulate functions of both RyR2 and full-length (FL) RyR3 by forming hetero-tetramers. Here, functional roles of RyR3 in the regulation of Ca2+ signaling in mesenteric artery SM cells (MASMCs) were examined using RyR3 homozygous knockout mice (RyR3-/-). Quantitative PCR analyses suggested that the predominant RyR3 subtype in MASMs from wild-type mice (RyR3+/+) was DN-RyR3. In single MASMCs freshly isolated from RyR3-/-, the EC50 of caffeine to induce Ca2+ release was lower than that in RyR3+/+ myocytes. The amplitude and frequency of Ca2+ sparks and spontaneous transient outward currents in MASMCs from RyR3-/- were all larger than those from RyR3+/+. Importantly, mRNA and functional expressions of voltage-dependent Ca2+ channel and large-conductance Ca2+-activated K+ (BK) channel in MASMCs from RyR3-/- were identical to those from RyR3+/+. However, in the presence of BK channel inhibitor, paxilline, the pressure rises induced by BayK8644 in MA vascular beds of RyR3-/- were significantly larger than in those of RyR3+/+. This indicates that the negative feedback effects of BK channel activity on intracellular Ca2+ signaling was enhanced in RyR3-/-. Thus, RyR3, and, in fact, mainly DN-RyR3, via a complex with RyR2 suppresses Ca2+ release and indirectly regulated membrane potential by reducing BK channel activity in MASMCs and presumably can affect the regulation of intrinsic vascular tone.