Calcium entry via TRPC6 mediates albumin overload-induced endoplasmic reticulum stress and apoptosis in podocytes

Albumin, which is the most abundant component of urine proteins, exerts injurious effects on renal cells in chronic kidney diseases. However, the toxicity of albumin to podocytes is not well elucidated. Here, we show that a high concentration of albumin triggers intracellular calcium ([Ca²⁺]_i) increase through mechanisms involving the intracellular calcium store release and extracellular calcium influx in conditionally immortalized podocytes. The canonical transient receptor potential-6 (TRPC6) channel, which is associated with a subset of familial forms of focal segmental glomerulosclerosis (FSGS) and several acquired proteinuric kidney diseases, was shown to be one of the important Ca²⁺ permeable ion channels in podocytes. Therefore we explored the role of TRPC6 on albumin-induced functional and structural changes in podocytes. It was found that albumin-induced increase in [Ca²⁺]_i was blocked by TRPC6 siRNA or SKF-96365, a blocker of TRP cation channels. Long-term albumin exposure caused an up-regulation of TRPC6 expression in podocytes, which was inhibited by TRPC6 siRNA. Additionally, the inhibition of TRPC6 prevented the F-actin cytoskeleton disruption that is induced by albumin overload. Moreover, albumin overload induced expression of the endoplasmic reticulum (ER) stress protein GRP78, led to caspase-12 activation and ultimately podocyte apoptosis, all of which were abolished by the knockdown of TRPC6 using TRPC6 siRNA. These results support the view that albumin overload may induce ER stress and the subsequent apoptosis in podocytes via TRPC6-mediated Ca²⁺ entry.     

Plasticity and emerging role of BKCa channels in nociceptive control in neuropathic pain

Large‐conductance Ca²⁺‐activated K⁺ (BK_Ca, MaxiK) channels are important for the regulation of neuronal excitability. Peripheral nerve injury causes plasticity of primary afferent neurons and spinal dorsal horn neurons, leading to central sensitization and neuropathic pain. However, little is known about changes in the BK_Ca channels in the dorsal root ganglion (DRG) and spinal dorsal horn and their role in the control of nociception in neuropathic pain. Here we show that L5 and L6 spinal nerve ligation in rats resulted in a substantial reduction in both the mRNA and protein levels of BK_Ca channels in the DRG but not in the spinal cord. Nerve injury primarily reduced the BK_Ca channel immunoreactivity in small‐ and medium‐sized DRG neurons. Furthermore, although the BK_Ca channel immunoreactivity was decreased in the lateral dorsal horn, there was an increase in the BK_Ca channel immunoreactivity present on dorsal horn neurons near the dorsal root entry zone. Blocking the BK_Ca channel with iberiotoxin at the spinal level significantly reduced the mechanical nociceptive withdrawal threshold in control and nerve‐injured rats. Intrathecal injection of the BK_Ca channel opener [1,3‐dihydro‐1‐[2‐hydroxy‐5‐(trifluoromethyl)phenyl]‐5‐(trifluoromethyl)‐2H‐benzimidazol‐2‐one] dose dependently reversed allodynia and hyperalgesia in nerve‐ligated rats but it had no significant effect on nociception in control rats. Our study provides novel information that nerve injury suppresses BK_Ca channel expression in the DRG and induces a redistribution of BK_Ca channels in the spinal dorsal horn. BK_Ca channels are increasingly involved in the control of sensory input in neuropathic pain and may represent a new target for neuropathic pain treatment.     

Activation of BKCa Channels Mediates Hippocampal Neuronal Death After Reoxygenation and Reperfusion

Excessive K⁺ efflux promotes central neuronal apoptosis; however, the type of potassium channel that mediates K⁺ efflux in response to different apoptosis-inducing stimuli is still unknown. It is hypothesized that the activation of large-conductance Ca²⁺-activated K⁺ channels (BK_Ca) mediates hypoxia/reoxygenation (H/R)- and ischemia/reperfusion (I/R)-induced neuronal apoptosis. Rat hippocampal neuronal cultures underwent apoptosis after reoxygenation, as assessed by morphologic observation, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and caspase-3 activation. Single-channel recordings revealed upregulation of BK_Ca channel activity 6 h after reoxygenation, which might be caused by elevated cytosolic Ca²⁺. The K⁺ ionophore valinomycin and the BK_Ca channel opener NS1619 induced neuronal apoptosis. Transfection of the BK_Ca channel α subunit into Chinese hamster ovary (CHO-K1) cells, which do not express endogenous K⁺ channels, or into neurons will induce cell apoptosis, indicating that the opening of the BK_Ca channel serves as a pivotal event in mediating cell apoptosis. The specific BK_Ca channel blockers charybdotoxin and iberiotoxin and the nonselective K⁺ channel blocker tetraethylammonium at concentrations more specific to the BK_Ca channel were neuroprotective. The A-type potassium channel blocker 4-aminopyridine and apamin, a small-conductance Ca²⁺-activated K⁺ channel blocker, were not protective. This result suggests the involvement of the BK_Ca channel in H/R-induced apoptosis. Similarly, specific BK_Ca channel blockers also showed neuroprotection in neurons subjected to oxygen-glucose deprivation/reoxygenation or animals subjected to forebrain ischemia–reperfusion. These results demonstrate that the over-activity of BK_Ca channels mediates hippocampal neuronal damage induced by H/R in vitro and I/R in vivo.     

多不饱和脂肪酸对钾通道的调控作用及机理

多不饱和脂肪酸具有包括离子通道在内的众多作用靶点,通过作用于这些靶点,可以有效保护免疫系统、神经系统和心血管系统的功能,在一定程度上保护人体健康。电压门控钾离子通道家族K_V7通道和大电导钙离子激活的钾离子通道(BK_Ca)广泛表达于机体的各类组织中,具有重要的生理或病理功能。本综述围绕K_V7和BK_Ca通道,根据对已有报道的汇总,多不饱和脂肪酸可以增大K_V7和BK_Ca通道的电流幅值,其中对K_V7通道电流的影响主要是改变其电压依赖特性和最大电导值,而对BK_Ca通道电流的影响主要是改变其孔道区域关闭态的构象。此外,多不饱和脂肪酸对K_V7和BK_Ca通道功能的调节也会受到共表达的辅助亚基影响,但相关机制有待进一步阐明。深入理解多不饱和脂肪酸对K_V7和BK_Ca通道调节作用效果和分子机制,有助于全面理解K_V7和BK     

BK Ca Channels Activating at Resting Potential without Calcium in LNCaP Prostate Cancer Cells

Large-conductance Ca²⁺-dependent K⁺ (BK_Ca) channels are activated by intracellular Ca²⁺ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK_Ca-type K⁺ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK_L. K⁺ selectivity, high conductance, activation by Mg²⁺ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK_Ca channels. However, unlike conventional BK_Ca channels, BK_L channels activated in the absence of free cytosolic Ca²⁺ at physiological membrane potentials; the half-maximal activation voltage was shifted by about −100 mV compared with BK_Ca channels. Half-maximal Ca²⁺-dependent activation was observed at 0.4 μM for BK_L (at −20 mV) and at 4.1 μM for BK_Ca channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK_L conductance. Expression of hSlo-β1 in LNCaP cells shifted voltage-dependent activation to values between that of BK_L and BK_Ca channels and reduced the slope of the P_open (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK_Ca subunit, which is responsible for the BK_L phenotype in a dominant manner. BK_L-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK_L in LNCaP cells is regulated by serum-derived factors, however not by androgens.     

Bupivacaine inhibits large conductance,voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K⁺ channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca²⁺-activated K⁺ channels (BK_Ca). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K⁺ currents carried by BK_Ca channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC₅₀ 324 µM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 µM) can also block whole-cell K⁺ currents (~45% blockage) in which, under our working conditions, BK_Ca is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK_Ca channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.     

Identification and characterization of Ca2+-activated K+ channels in granulosa cells of the human ovary

Background  

Granulosa cells (GCs) represent a major endocrine compartment of the ovary producing sex steroid hormones. Recently, we identified in human GCs a Ca²⁺-activated K⁺ channel (K_Ca) of big conductance (BK_Ca), which is involved in steroidogenesis. This channel is activated by intraovarian signalling molecules (e.g. acetylcholine) via raised intracellular Ca²⁺ levels. In this study, we aimed at characterizing 1. expression and functions of K_Ca channels (including BK_Ca beta-subunits), and 2. biophysical properties of BK_Ca channels.     

Activation of cloned BK<Subscript>Ca</Subscript> channels in nitric oxide-induced apoptosis of HEK293 cells

The large conductance Ca²⁺-activated K⁺ (BK_Ca) channels are highly expressed in vascular smooth muscle cells (VSMCs) and play an essential role in the regulation of various physiological functions. Besides its electrophysiological function in vascular relaxation, BK_Ca has also been reported to be implicated in nitric oxide (NO)-induced apoptosis of VSMCs. However, the molecular mechanism is not clear and has not been determined on cloned channels. The present study was designed to clarify whether activation of cloned BK_Ca channel was involved in NO-induced apoptosis in human embryonic kidney 293 (HEK293) cell. The cDNA encoding the α-subunit of BK_Ca channel, hSloα, was transiently transfected into HEK293 cells. The apoptotic death in HEK-hSloα cells was detected using immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, MTT test, and flow cytometry assays. Whole-cell and single-channel characteristics of HEK-hSloα cells exhibited functional features similar to native BK_Ca channel in VSMCs. Exposuring of HEK- hSloα cells to S-nitroso-N-acetyl-penicillamine increased the hSloα channel activities of whole-cell and single-channel, and then increased percentage of cells undergoing apoptosis. However, blocking hSloα channels with 1 mM tetraethylammonia or 100 nM iberiotoxin significantly decreased the NO-induced apoptosis, whereas 30 μM NS1619, the specific agonist of BK_Ca, independently increased hSloα currents and induced apoptosis. These results indicated that activation of cloned BK_Ca channel was involved in NO-induced apoptosis of HEK293 cells.     

Modeling a Ca2+ Channel/BKCa Channel Complex at the Single-Complex Level

BK_Ca-channel activity often affects the firing properties of neurons, the shapes of neuronal action potentials (APs), and in some cases the extent of neurotransmitter release. It has become clear that BK_Ca channels often form complexes with voltage-gated Ca²⁺ channels (CaV channels) such that when a CaV channel is activated, the ensuing influx of Ca²⁺ activates its closely associated BK_Ca channel. Thus, in modeling the electrical properties of neurons, it would be useful to have quantitative models of CaV/BK_Ca complexes. Furthermore, in a population of CaV/BK_Ca complexes, all BK_Ca channels are not exposed to the same Ca²⁺ concentration at the same time. Thus, stochastic rather than deterministic models are required. To date, however, no such models have been described. Here, however, I present a stochastic model of a CaV2.1/BK_Ca(α-only) complex, as might be found in a central nerve terminal. The CaV2.1/BK_Ca model is based on kinetic modeling of its two component channels at physiological temperature. Surprisingly, The CaV2.1/BK_Ca model predicts that although the CaV channel will open nearly every time during a typical cortical AP, its associated BK_Ca channel is expected to open in only 30% of trials, and this percentage is very sensitive to the duration of the AP, the distance between the two channels in the complex, and the presence of fast internal Ca²⁺ buffers. Also, the model predicts that the kinetics of the BK_Ca currents of a population of CaV2.1/BK_Ca complexes will not be limited by the kinetics of the CaV2.1 channel, and during a train of APs, the current response of the complex is expected to faithfully follow even very rapid trains. Aside from providing insight into how these complexes are likely to behave in vivo, the models presented here could also be of use more generally as components of higher-level models of neural function.     

CaV1.3 L-type channels,maxiK Ca2 +-dependent K+ channels and bestrophin-1 regulate rhythmic photoreceptor outer segment phagocytosis by retinal pigment epithelial cells

Phagocytosis of shed photoreceptor outer segments by the retinal pigment epithelium (RPE) is critical for maintenance of visual function. Because changes in intracellular Ca^2 + regulate phagocytosis, we studied in vitro the impact of different ion channels in addition to mice deficient for Ca_v1.3 L-type Ca²⁺ channels (Ca1.3^−/−) and maxiK Ca²⁺-dependent K⁺ channels (BK^−/−). The knockdown of Bestrophin-1 protein, a regulator of intracellular Ca²⁺ homeostasis, affected phagocytosis in porcine RPE cultures. Blockage of voltage-gated L-type channels by (+)BayK8644 inhibitor reduced phagocytosis in vitro, in contrast L-type activation by (−)BayK8644 had no impact. The expression rate of Ca_v1.3, the predominant L-type Ca^2 + channel in RPE cells, varied at different times of day. Ca_V1.3^−/− RPE lacked peak phagocytic activity following morning photoreceptor shedding in wild-type RPE and retained a higher number of phagosomes at a later time of day. The BK-channel blocker paxilline lowered phagocytosis in RPE cultures in a concentration-dependent manner. BK^−/− RPE in vivo retained phagocytic capability but this activity, which is normally well synchronized with circadian photoreceptor shedding, shifted out of phase. Retinae of older BK^−/− mice showed shortened photoreceptor outer segments and diminished rhodopsin content. Store-operated Ca^2 + channels Orai-1 did not affect phagocytosis in cultured RPE. TRPV channel inhibition by ruthenium-red reduced phagocytosis, whereas activation at high concentrations of 2-APB increased phagocytosis. Our data demonstrate essential roles for bestrophin-1, BK, TRPV and L-type channels in regulating retinal phagocytosis. These data indicate further the importance of BK and Ca_V1.3 for rhythmic phagocytic activity synchronized with photoreceptor shedding.     

Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

Plasma membrane large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BK_Ca channel regulation by IP₃ and IP₃Rs in rat and mouse cerebral artery smooth muscle cells. IP₃ activated BK_Ca channels both in intact cells and in excised inside-out membrane patches. IP₃ caused concentration-dependent BK_Ca channel activation with an apparent dissociation constant (K_d) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca²⁺ concentration ([Ca²⁺]_i; 10 µM). IP₃ also caused a leftward-shift in BK_Ca channel apparent Ca²⁺ sensitivity and reduced the K_d for free [Ca²⁺]_i from ∼20 to 12 µM, but did not alter the slope or maximal P_o. BAPTA, a fast Ca²⁺ buffer, or an elevation in extracellular Ca²⁺ concentration did not alter IP₃-induced BK_Ca channel activation. Heparin, an IP₃R inhibitor, and a monoclonal type 1 IP₃R (IP₃R1) antibody blocked IP₃-induced BK_Ca channel activation. Adenophostin A, an IP₃R agonist, also activated BK_Ca channels. IP₃ activated BK_Ca channels in inside-out patches from wild-type (IP₃R1^+/+) mouse arterial smooth muscle cells, but had no effect on BK_Ca channels of IP₃R1-deficient (IP₃R1^−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP₃R1 is located in close spatial proximity to BK_Ca α subunits. The IP₃R1 monoclonal antibody coimmunoprecipitated IP₃R1 and BK_Ca channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP₃R1 activation elevates BK_Ca channel apparent Ca²⁺ sensitivity through local molecular coupling in arterial smooth muscle cells.     

Mechanosensitivity of mitochondrial large-conductance calcium-activated potassium channels

Potassium channels have been discovered in the inner mitochondrial membrane of various cells. These channels can regulate the mitochondrial membrane potential, the matrix volume, respiration and reactive species generation. Therefore, it is believed that their activation is cytoprotective in various tissues. In our study, the single-channel activity of a large-conductance calcium-activated potassium channel (mitoBK_Ca) was measured by the patch-clamp technique on mitoplasts derived from mitochondria isolated from human glioma U-87 MG cells. Here, we show for the first time that mechanical stimulation of mitoBK_Ca channels results in an increased probability of channel opening. However, the mechanosensitivity of mitoBK_Ca channels was variable with some channels exhibiting no mechanosensitivity. We detected the expression of mechanosensitive BK_Ca-STREX exon in U-87 MG cells and hypotesize, based on previous studies demonstrating the presence of multiple BK_Ca splice variants that variable mechanosensitivity of mitoBK_Ca could be the result of the presence of diverse BK_Ca isoforms in mitochondria of U-87 MG cells. Our findings indicate the possible involvement of the mitoBK_Ca channel in mitochondria activities in which changes in membrane tension and shape play a crucial role, such as fusion/fission and cristae remodeling.     

IP3 decreases coronary artery tone via activating the BKCa channel of coronary artery smooth muscle cells in pigs

Large conductance Ca²⁺-activated K⁺ channel (BK_Ca) is a potential target for coronary artery-relaxing medication, but its functional regulation is largely unknown. Here, we report that inositol trisphosphate (IP3) activated BK_Ca channels in isolated porcine coronary artery smooth muscle cells and by which decreased the coronary artery tone. Both endogenous and exogenous IP3 increased the spontaneous transient outward K⁺ currents (STOC, a component pattern of BK_Ca currents) in perforated and regular whole-cell recordings, which was dependent on the activity of IP3 receptors. IP3 also increased the macroscopic currents (MC, another component pattern of BK_Ca currents) via an IP3 receptor- and sarcoplasmic Ca²⁺ mobilization-independent pathway. In inside-out patch recordings, direct application of IP3 to the cytosolic side increased the open probability of single BK_Ca channel in an IP3 receptor-independent manner. We conclude that IP3 is an activator of BK_Ca channels in porcine coronary smooth muscle cells and exerts a coronary artery-relaxing effect. The activation of BK_Ca channels by IP3 involves the enhancement of STOCs via IP3 receptors and stimulation of MC by increasing the Ca²⁺ sensitivity of the channels.     

Functional and molecular consequences of ionizing irradiation on large conductance Ca2+-activated K+ channels in rat aortic smooth muscle cells

AimsThe goal of this study was to evaluate the influence of γ-irradiation on Ca²⁺-activated K⁺ channel (BK_Ca) function and expression in rat thoracic aorta.Main methodsAortic cells or tissues were studied by the measurement of force versus [Ca²⁺]_i, patch-clamp technique, and RT-PCR.Key findingsStimulation of smooth muscle cells with depolarizing voltage steps showed expression of outward K⁺ currents. Paxilline, an inhibitor of BK_Ca channels, decreased outward K⁺ current density. Outward currents in smooth muscle cells obtained from irradiated animals 9 and 30 days following radiation exposure demonstrated a significant decrease in K⁺ current density. Paxilline decreased K⁺ current in cells obtained 9 days, but was without effect 30 days after irradiation suggesting the absence of BK_Ca channels. Aortic tissue from irradiated animals showed progressively enhanced contractile responses to phenylephrine in the post-irradiation period of 9 and 30 days. The concomitant Ca²⁺ transients were significantly smaller, as compared to tissues from control animals, 9 days following irradiation but were increased above control levels 30 days following irradiation. Irradiation produced a decrease in BK_Ca α- and β₁-subunit mRNA levels in aortic smooth muscle cells suggesting that the vasorelaxant effect of these channels may be diminished.SignificanceThese results suggest that the enhanced contractility of vascular tissue from animals exposed to radiation may result from an increase in myofilament Ca²⁺ sensitivity in the early post-irradiation period and a decrease in BK_Ca channel expression in the late post-irradiation period.     

Potentiation of large-conductance calcium-activated potassium (BK(Ca)) channels by a specific isoform of protein kinase C

The phosphorylation state of large-conductance calcium-activated potassium (BK_Ca) channels regulates their activity and is dynamically regulated by protein phosphatases and kinases, including protein kinase C (PKC). In this study, we showed that PKC activators up-regulate the activity of the BK_Ca channel alpha (α)-subunit, Slo1, in cell-attached patches of transfected COS7 cells. In an immune complex kinase assay, BK_Ca channels isolated from rat brain were phosphorylated in the presence of PKC activators, without the addition of exogenous PKC, which suggests that PKC and BK_Ca channels functionally interact in vivo. Four different PKC isozymes, including PKCδ, phosphorylated the C-terminus of Slo1 and the addition of purified PKCδ-activated BK_Ca channels in excised patches of transfected HEK293 cells. Our results demonstrate that PKC up-regulates BK_Ca channels and that PKCδ may functionally interact with BK_Ca channel complexes in vivo.     

Contribution of BKCa-Channel Activity in Human Cardiac Fibroblasts to Electrical Coupling of Cardiomyocytes-Fibroblasts

Cardiac fibroblasts are involved in the maintenance of myocardial tissue structure. However, little is known about ion currents in human cardiac fibroblasts. It has been recently reported that cardiac fibroblasts can interact electrically with cardiomyocytes through gap junctions. Ca²⁺-activated K⁺ currents (I _K[Ca]) of cultured human cardiac fibroblasts were characterized in this study. In whole-cell configuration, depolarizing pulses evoked I _K(Ca) in an outward rectification in these cells, the amplitude of which was suppressed by paxilline (1 μM) or iberiotoxin (200 nM). A large-conductance, Ca²⁺-activated K⁺ (BK_Ca) channel with single-channel conductance of 162 ± 8 pS was also observed in human cardiac fibroblasts. Western blot analysis revealed the presence of α-subunit of BK_Ca channels. The dynamic Luo-Rudy model was applied to predict cell behavior during direct electrical coupling of cardiomyocytes and cardiac fibroblasts. In the simulation, electrically coupled cardiac fibroblasts also exhibited action potential; however, they were electrically inert with no gap-junctional coupling. The simulation predicts that changes in gap junction coupling conductance can influence the configuration of cardiac action potential and cardiomyocyte excitability. I _k(Ca) can be elicited by simulated action potential waveforms of cardiac fibroblasts when they are electrically coupled to cardiomyocytes. This study demonstrates that a BK_Ca channel is functionally expressed in human cardiac fibroblasts. The activity of these BK_Ca channels present in human cardiac fibroblasts may contribute to the functional activities of heart cells through transfer of electrical signals between these two cell types.     

Role of Voltage-Gated K+ (KV) Channels in Vascular Function

Voltage-dependent K⁺ (K_V) channels represent the most diverse group of K⁺ channels ubiquitously expressed in vascular smooth muscles. The K_V channels, together with other types of K⁺ conductances, such as Ca²⁺-activated (BK_Ca), ATP-sensitive (K_ATP), and inward rectifier, play an important role in the control of the cell membrane potential and regulation of the vascular contractility. Comparison of the expression of different K_V channel isoforms obtained from RT-PCR studies showed that virtually all K_V genes could be detected in vascular smooth muscle cells (VSMC). Based on the analysis of both mRNA and protein expressions, it is likely that K_V1.1, K_V1.2, K_V1.3, K_V1.5, K_V1.6, K_V2.1, and K_V3.1b channel isoforms are mainly responsible for the delayed rectifier current characterized electrophysiologically in most VSMC types studied to date. It has been recently demonstrated by our research group and by others that functional expression of multiple K_V channel α-subunits is not homogeneous and varies in different vascular beds of small and large arteries. Growing evidence suggests that in some small arteries, e.g., cerebral arteries and arterioles, the K_V channels are activated at more negative membrane voltages than BK_Ca, thus making a greater contribution to the control of vascular tone. Our data also suggest that in some blood vessels, such as the rat aorta and mouse small mesenteric arteries, the K_V channel current (identified mainly as passed through K_V2.1 channels), but not BK_Ca, is the predominant conductance activated even under conditions where intracellular Ca₂₊ concentration is increased up to 200 nM. In addition, our data indicate that the K_V2.1 channel current could also contribute to the regulation of the induced rhythmic activity in the rat aorta in vitro acting as a negative feedback mechanism for membrane depolarization. We and other experimenters also demonstrated that functional expression of K_V channels is a dynamic process, which is altered under normal physiological conditions (e.g., during the development of the vessels), and in various pathological states (e.g., pulmonary hypertension developing during chronic hypoxia). Recent findings also suggest that activation of K_V channels can also play a role in vascular apoptosis (causing loss of intracellular K⁺ and subsequent cell shrinking, one of the essential prerequisites of cellular apoptosis). To summarize, the K^V channels are essential for normal vascular function, and their expression and properties are altered under abnormal conditions. Therefore, understanding of the molecular identity of native K_V channels and their functional significance and elucidation of the mechanisms, which govern and control the expression of the K_V channels in the vasculature, represent an important and challenging task and could also lead to the development of useful therapeutic strategies for the treatment of cardiovascular diseases.     

Exercise training changes the gating properties of large-conductance Ca2+-activated K+ channels in rat thoracic aorta smooth muscle cells

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels play a critical role in regulating the cellular excitability in response to change in blood flow. It has been demonstrated that vascular BK_Ca channel currents in both humans and rats are increased after exercise training. This up-regulation of the BK_Ca channel activity in arterial myocytes may represent a cellular compensatory mechanism of limiting vascular reactivity to exercise training. However, the underlying mechanisms are not fully understood. In the present study, we examined the single channel activities and kinetics of the BK_Ca channels in rat thoracic aorta smooth muscle cells. We showed that exercise training significantly increased the open probability (Po), decreased the mean closed time and increased the mean open time, and the sensitivity to Ca²⁺ and voltage without altering the unitary conductance and the K⁺ selectivity. Our results suggest a novel mechanism by which exercise training increases the K⁺ currents by changing the BK_Ca channel activities and kinetics.     

Effects of BKCa and Kir2.1 Channels on Cell Cycling Progression and Migration in Human Cardiac c-kit+ Progenitor Cells

Our previous study demonstrated that a large-conductance Ca²⁺-activated K⁺ current (BK_Ca), a voltage-gated TTX-sensitive sodium current (I_Na.TTX), and an inward rectifier K⁺ current (I_Kir) were heterogeneously present in most of human cardiac c-kit⁺ progenitor cells. The present study was designed to investigate the effects of these ion channels on cell cycling progression and migration of human cardiac c-kit⁺ progenitor cells with approaches of cell proliferation and mobility assays, siRNA, RT-PCR, Western blots, flow cytometry analysis, etc. It was found that inhibition of BK_Ca with paxilline, but not I_Na.TTX with tetrodotoxin, decreased both cell proliferation and migration. Inhibition of I_Kir with Ba²⁺ had no effect on cell proliferation, while enhanced cell mobility. Silencing KCa.1.1 reduced cell proliferation by accumulating the cells at G0/G1 phase and decreased cell mobility. Interestingly, silencing Kir2.1 increased the cell migration without affecting cell cycling progression. These results demonstrate the novel information that blockade or silence of BK_Ca channels, but not I_Na.TTX channels, decreases cell cycling progression and mobility, whereas inhibition of Kir2.1 channels increases cell mobility without affecting cell cycling progression in human cardiac c-kit⁺ progenitor cells.