Large Conductance Calcium Research Articles

A computational model of large conductance voltage and calcium activated potassium channels: implications for calcium dynamics and electrophysiology in detrusor smooth muscle cells.

The large conductance voltage and calcium activated potassium (BK) channels play a crucial role in regulating the excitability of detrusor smooth muscle, which lines the wall of the urinary bladder. These channels have been widely characterized in terms of their molecular structure, pharmacology and electrophysiology. They control the repolarising and hyperpolarising phases of the action potential, thereby regulating the firing frequency and contraction profiles of the smooth muscle. Several groups have reported varied profiles of BK currents and I-V curves under similar experimental conditions. However, no single computational model has been able to reconcile these apparent discrepancies. In view of the channels' physiological importance, it is imperative to understand their mechanistic underpinnings so that a realistic model can be created. This paper presents a computational model of the BK channel, based on the Hodgkin-Huxley formalism, constructed by utilising three activation processes - membrane potential, calcium inflow from voltage-gated calcium channels on the membrane and calcium released from the ryanodine receptors present on the sarcoplasmic reticulum. In our model, we attribute the discrepant profiles to the underlying cytosolic calcium received by the channel during its activation. The model enables us to make heuristic predictions regarding the nature of the sub-membrane calcium dynamics underlying the BK channel's activation. We have employed the model to reproduce various physiological characteristics of the channel and found the simulated responses to be in accordance with the experimental findings. Additionally, we have used the model to investigate the role of this channel in electrophysiological signals, such as the action potential and spontaneous transient hyperpolarisations. Furthermore, the clinical effects of BK channel openers, mallotoxin and NS19504, were simulated for the detrusor smooth muscle cells. Our findings support the proposed application of these drugs for amelioration of the condition of overactive bladder. We thus propose a physiologically realistic BK channel model which can be integrated with other biophysical mechanisms such as ion channels, pumps and exchangers to further elucidate its micro-domain interaction with the intracellular calcium environment.

Expression and Activation of BKCa Channels in Mice Protects Against Ischemia-Reperfusion Injury of Isolated Hearts by Modulating Mitochondrial Function.

Aims: Activation and expression of large conductance calcium and voltage-activated potassium channel (BKCa) by pharmacological agents have been implicated in cardioprotection from ischemia-reperfusion (IR) injury possibly by regulating mitochondrial function. Given the non-specific effects of pharmacological agents, it is not clear whether activation of BKCa is critical to cardioprotection. In this study, we aimed to decipher the mechanistic role of BKCa in cardioprotection from IR injury by genetically activating BKCa channels.Methods and Results: Hearts from adult (3 months old) wild-type mice (C57/BL6) and mice expressing genetically activated BKCa (Tg-BKCaR207Q, referred as Tg-BKCa) along with wild-type BKCa were subjected to 20 min of ischemia and 30 min of reperfusion with or without ischemic preconditioning (IPC, 2 times for 2.5 min interval each). Left ventricular developed pressure (LVDP) was recorded using Millar's Mikrotip® catheter connected to ADInstrument data acquisition system. Myocardial infarction was quantified by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Our results demonstrated that Tg-BKCa mice are protected from IR injury, and BKCa also contributes to IPC-mediated cardioprotection. Cardiac function parameters were also measured by echocardiography and no differences were observed in left ventricular ejection fraction, fractional shortening and aortic velocities. Amplex Red® was used to assess reactive oxygen species (ROS) production in isolated mitochondria by spectrofluorometry. We found that genetic activation of BKCa reduces ROS after IR stress. Adult cardiomyocytes and mitochondria from Tg-BKCa mice were isolated and labeled with Anti-BKCa antibodies. Images acquired via confocal microscopy revealed localization of cardiac BKCa in the mitochondria.Conclusions: Activation of BKCa is essential for recovery of cardiac function after IR injury and is likely a factor in IPC mediated cardioprotection. Genetic activation of BKCa reduces ROS produced by complex I and complex II/III in Tg-BKCa mice after IR, and IPC further decreases it. These results implicate BKCa-mediated cardioprotection, in part, by reducing mitochondrial ROS production. Localization of Tg-BKCa in adult cardiomyocytes of transgenic mice was similar to BKCa in wild-type mice.

Propofol-Induced Vasodilation in Human Internal Mammary Artery: Role of Potassium Channels

The aim of this study was to investigate the vascular effects and mechanisms of propofol in the human internal mammary artery (IMA). In vitro experimental study. The study was conducted inthe research laboratory of a pharmacology department. IMA segments were obtained from 52 patients undergoing coronary artery bypass surgery. The IMA rings were suspended in isolated organ baths, and the changes in the tension were isometrically recorded. The antagonistic effect of propofol (1 μM, 10 μM, and 100 μM) on contractions induced by potassium chloride (45 mM), phenylephrine (1 μM), 5-hydroxytryptamine (30 μM), and calcium chloride (10 μM-10 mM) was investigated. The relaxations induced by propofol also were tested in the presence of the nitric oxide synthase inhibitor, nitro-L-arginine methyl ester (100 mM); the cyclooxygenase inhibitor, indomethacin (10 mM); and the potassium ion channel inhibitors, tetraethylammonium (1 mM), iberiotoxin (20 nM), glibenclamide (10 µM), 4-aminopyridine (1 mM), and barium chloride (30 µM). Propofol caused a significant concentration-dependent vasorelaxation, which was endothelium independent. It inhibited the contractions induced by potassium chloride, phenylephrine, 5-hydroxytryptamine, and calcium chloride (p < 0.001), but it did not affect the basal tension. Propofol-induced relaxation was significantly inhibited by iberiotoxin and tetraethylammonium (p < 0.001); however, it was not affected by 4-aminopyridine, glibenclamide, and barium chloride. This study clearly reveals that propofol relaxes the IMA, and propofol-induced vasodilation may be related to large conductance calcium ion-activated potassium ion channel activation. Propofol use in coronary artery bypass surgery can be valuable via its favorable vasodilator effect to overcome perioperative vasospasm of IMA.

Overview of Bile Acids Signaling and Perspective on the Signal of Ursodeoxycholic Acid, the Most Hydrophilic Bile Acid, in the Heart.

Bile acids (BA) are classically known as an important agent in lipid absorption and cholesterol metabolism. Nowadays, their role in glucose regulation and energy homeostasis are widely reported. BAs are involved in various cellular signaling pathways, such as protein kinase cascades, cyclic AMP (cAMP) synthesis, and calcium mobilization. They are ligands for several nuclear hormone receptors, including farnesoid X-receptor (FXR). Recently, BAs have been shown to bind to muscarinic receptor and Takeda G-protein-coupled receptor 5 (TGR5), both G-protein-coupled receptor (GPCR), independent of the nuclear hormone receptors. Moreover, BA signals have also been elucidated in other nonclassical BA pathways, such as sphingosine-1-posphate and BK (large conductance calcium- and voltage activated potassium) channels. Hydrophobic BAs have been proven to affect heart rate and its contraction. Elevated BAs are associated with arrhythmias in adults and fetal heart, and altered ratios of primary and secondary bile acid are reported in chronic heart failure patients. Meanwhile, in patients with liver cirrhosis, cardiac dysfunction has been strongly linked to the increase in serum bile acid concentrations. In contrast, the most hydrophilic BA, known as ursodeoxycholic acid (UDCA), has been found to be beneficial in improving peripheral blood flow in chronic heart failure patients and in protecting the heart against reperfusion injury. This review provides an overview of BA signaling, with the main emphasis on past and present perspectives on UDCA signals in the heart.

Glutamate-activated BK channel complexes formed with NMDA receptors

The large-conductance calcium- and voltage-activated K+ (BK) channel has a requirement of high intracellular free Ca2+ concentrations for its activation in neurons under physiological conditions. The Ca2+ sources for BK channel activation are not well understood. In this study, we showed by coimmunopurification and colocalization analyses that BK channels form complexes with NMDA receptors (NMDARs) in both rodent brains and a heterologous expression system. The BK-NMDAR complexes are broadly present in different brain regions. The complex formation occurs between the obligatory BKα and GluN1 subunits likely via a direct physical interaction of the former's intracellular S0-S1 loop with the latter's cytosolic regions. By patch-clamp recording on mouse brain slices, we observed BK channel activation by NMDAR-mediated Ca2+ influx in dentate gyrus granule cells. BK channels modulate excitatory synaptic transmission via functional coupling with NMDARs at postsynaptic sites of medial perforant path-dentate gyrus granule cell synapses. A synthesized peptide of the BKα S0-S1 loop region, when loaded intracellularly via recording pipette, abolished the NMDAR-mediated BK channel activation and effect on synaptic transmission. These findings reveal the broad expression of the BK-NMDAR complexes in brain, the potential mechanism underlying the complex formation, and the NMDAR-mediated activation and function of postsynaptic BK channels in neurons.

Impaired Trafficking of β1 Subunits Inhibits BK Channels in Cerebral Arteries of Hypertensive Rats.

Hypertension is a risk factor for cerebrovascular diseases, including stroke and dementia. During hypertension, arteries become constricted and are less responsive to vasodilators, including nitric oxide (NO). The regulation of arterial contractility by smooth muscle cell (myocyte) large-conductance calcium (Ca2+)-activated potassium (BK) channels is altered during hypertension, although mechanisms involved are unclear. We tested the hypothesis that dysfunctional trafficking of pore-forming BK channel (BKα) and auxiliary β1 subunits contributes to changes in cerebral artery contractility of stroke-prone spontaneously hypertensive rats (SP-SHRs). Our data indicate that the amounts of total and surface BKα and β1 proteins are similar in unstimulated arteries of age-matched SP-SHRs and normotensive Wistar-Kyoto rats. In contrast, stimulated surface-trafficking of β1 subunits by NO or membrane depolarization is inhibited in SP-SHR myocytes. PKCα (protein kinase C α) and PKCβII total protein and activity were both higher in SP-SHR than in Wistar-Kyoto rat arteries. NO or depolarization robustly activated Rab11, a small trafficking GTPase, in Wistar-Kyoto rat arteries but weakly activated Rab11 in SP-SHRs. Bisindolylmaleimide, a PKC inhibitor, and overexpression of a PKC phosphorylation-deficient Rab11A mutant (Rab11A S177A) restored stimulated β1 subunit surface-trafficking in SP-SHR myocytes. BK channel activation by NO was inhibited in SP-SHR myocytes and restored by Rab11A S177A expression. Vasodilation to NO and lithocholate, a BKα/β1 channel activator, was inhibited in pressurized SP-SHR arteries and reestablished by bisindolylmaleimide. In summary, data indicate that spontaneously active PKC inhibits Rab11A-mediated β1 subunit trafficking in arterial myocytes of SP-SHRs, leading to dysfunctional NO-induced BK channel activation and vasodilation.

Pathophysiology in the suprachiasmatic nucleus in mouse models of Huntington's disease.

Disturbances in sleep/wake cycle are a common complaint of individuals with Huntington's disease (HD) and are displayed by HD mouse models. The underlying mechanisms, including the possible role of the circadian timing system, are not well established. The BACHD mouse model of HD exhibits disrupted behavioral and physiological rhythms, including decreased electrical activity in the central circadian clock (suprachiasmatic nucleus, SCN). In this study, electrophysiological techniques were used to explore the ionic underpinning of the reduced spontaneous neural activity in male mice. We found that SCN neural activity rhythms were lost early in the disease progression and was accompanied by loss of the normal daily variation in resting membrane potential in the mutant SCN neurons. The low neural activity could be transiently reversed by direct current injection or application of exogenous N-methyl-d-aspartate (NMDA) thus demonstrating that the neurons have the capacity to discharge at WT levels. Exploring the potassium currents known to regulate the electrical activity of SCN neurons, our most striking finding was that these cells in the mutants exhibited an enhancement in the large-conductance calcium activated K+ (BK) currents. The expression of the pore forming subunit (Kcnma1) of the BK channel was higher in the mutant SCN. We found a similar decrease in daytime electrical activity and enhancement in the magnitude of the BK currents early in disease in another HD mouse model (Q175). These findings suggest that SCN neurons of both HD models exhibit early pathophysiology and that dysregulation of BK current may be responsible.

Chronic exercise impairs nitric oxide pathway in rabbit carotid and femoral arteries.

Some of the beneficial effects of exercise in preventing vascular related diseases are mediated by the enhancement of endothelial function where the role of nitric oxide (NO) is well documented, although the relevance of calcium activated potassium channels is not fully understood. The impact of oxidative stress induced by training on endothelial function remains to be clarified. By evaluating different endothelial vasodilator pathways on two vascular beds in a rabbit model of chronic exercise, we found a decreased NO bioavailability and endothelial nitric oxide synthase expression in both carotid and femoral arteries. Physical training induced carotid endothelial dysfunction as a result of an increase in oxidative stress and a reduction in superoxide dismutase expression. In the femoral artery, the lower production of NO was counteracted by an increased participation of large conductance calcium activated potassium channels, preventing endothelial dysfunction. The present study aimed to evaluate the effects of chronic exercise on vasodilator response in two different arteries. Rings of carotid and femoral arteries from control and trained rabbits were suspended in organ baths for isometric recording of tension. Endothelial nitric oxide synthase (eNOS), Cu/Zn and Mn-superoxide dismutase (SOD), and large conductance calcium activated potassium (BKCa) channel protein expression were measured by western blotting. In the carotid artery, training reduced the relaxation to ACh (10-9 to 3×10-6 m) that was reversed by N-acetylcysteine (10-3 m). l-NAME (10-4 m) reduced the relaxation to ACh in both groups, although the effect was lower in the trained group (in mean ± SEM, 39±2% vs. 28±3%). Physical training did not modify the relaxation to ACh in femoral arteries, although the response to l-NAME was lower in the trained group (in mean ± SEM, 41±5% vs. 17±2%). Charybdotoxin (10-7 m) plus apamin (10-6 m) further reduced the maximal relaxation to ACh only in the trained group. The remaining relaxation in both carotid and femoral arteries was abolished by KCl (2×10-2 m) and BaCl2 (3×10-6 m) plus ouabain (10-4 m) in both groups. Physical training decreased eNOS expression in both carotid and femoral arteries and Cu/Zn and Mn-SOD expression only in the carotid artery. BKCa channels were overexpressed in the trained group in the femoral artery. In conclusion, chronic exercise induces endothelial dysfunction in the carotid artery as a result of oxidative stress. In the femoral artery, it modifies the vasodilator pathways, enhancing the participation of BKCa channels, thus compensating for the impairment of NO-mediated vasodilatation.

Propofol-induced vasodilation of mesenteric arterioles via BKCa channel and gap junction.

The present study aimed to investigate the role of propofol in mediating the vasomotor activity of the mesenteric arteriole (MA) of Sprague Dawley (SD) rats, and to elucidate the underlying mechanisms. The pressure myograph technique was used to examine the effect of different concentrations of propofol on the relaxation of blood vessels in the 2–3 mm MA segments freshly separated from the SD rats. The whole-cell patch-clamp technique was applied to observe the outward current of single vascular smooth muscle cells (VSMCs) obtained from the MAs of the SD rats. Furthermore, immunofluorescence was utilized to assess the expression of connexin (Cx) in the MAs of SD rats. The results indicated the following: i) Propofol relaxed the MA of SD rats in a concentration-dependent manner from 1×10−7 to 3×10−4 mol/l; ii) in the acutely dissociated VSMCs, propofol (1×10−7 to 3×10−4 mol/l) enhanced the outward current of VSMCs in a concentration-dependent manner; iii) the enhanced outward currents induced by propofol (1×10−5 mol/l) may be reversed by tetraethylammonium (TEA; 1 mmol/l), a calcium-activated K+ channel inhibitor; iv) the effect of propofol on the relaxation of the vasculature wAS reduced after perfusion with 1 mmol/l TEA; v) Cx40, Cx43 and Cx45 were expressed on the MA; 6) 18β-glycyrrhetintic acid and 2-aminoethoxydiphenyl borate, two types of gap junction blocker, inhibited the propofol-induced relaxation. The present study provides evidence that propofol relaxes the MA, which may be associated with its effect of enhancing the channel current of large-conductance calcium voltage-activated potassium channels, contributing to the K+ outflow and leading to VSMC hyperpolarization; the gap junction may facilitate the hyperpolarization, which may lead to vascular synchronized relaxation and thereby reduce the blood pressure.

The Large Conductance Calcium- and Voltage-activated Potassium Channel (BK) and Epilepsy.

The large conductance, calcium- and voltage-activated potassium channels (BK) are widely distributed channel proteins which exist in virtually every cell type of mammals and function to influence membrane excitability and Ca2+ signaling. BK channels can be activated by the increase of the intracellular Ca2+ concentration, a consequence of neuronal excitation, and then terminate the action potential with the outward K+ flux. Moreover, after-hyperpolarization induced by BK channels closes Cav channels and thus precludes excessive Ca2+ influx. Considering this negative feedback effect, BK channel seemly acts to decrease membrane excitability in order to prevent hyperexcitation which is a typical characteristic of epilepsy. Therefore, one may reasonably suppose that membrane excitability would increase when the BK channel activity decreases. However, the membrane excitability displays elevation when the function of BK channel is under either upregulated or down-regulated status. Factors altering the activity of BK channels, such as gene mutations, polymorphism, channel openers or blockers that lead to loss- or gain-of-function, have all been linked to epilepsy onset. The aim of this review is to summarize existing knowledge and recent findings on the molecular properties, signaling complex and channel dysfunction of the BK channels with a particular attention to the possible relevance to the pathophysiology of epilepsy.

Inhibition of BKCa negatively alters cardiovascular function.

Large conductance calcium and voltage‐activated potassium channels (BKC a) are transmembrane proteins, ubiquitously expressed in the majority of organs, and play an active role in regulating cellular physiology. In the heart, BKC a channels are known to play a role in regulating the heart rate and protect it from ischemia–reperfusion injury. In vascular smooth muscle cells, the opening of BKC a channels results in membrane hyperpolarization which eventually results in vasodilation mediated by a reduction in Ca2+ influx due to the closure of voltage‐dependent Ca2+ channels. Ex vivo studies have shown that BKC a channels play an active role in the regulation of the function of the majority of blood vessels. However, in vivo role of BKC a channels in cardiovascular function is not completely deciphered. Here, we have evaluated the rapid in vivo role of BKC a channels in regulating the cardiovascular function by using two well‐established, rapid‐acting, potent blockers, paxilline and iberiotoxin. Our results show that BKC a channels are actively involved in regulating the heart rate, the function of the left and right heart as well as major vessels. We also found that the effect on BKC a channels by blockers is completely reversible, and hence, BKC a channels can be exploited as potential targets for clinical applications for modulating heart rate and cardiac contractility.

The role of BKCa in endometrial cancer HEC-1-B cell proliferation and migration

BKCa is a large conductance calcium activated potassium channel ubiquitously expressed in various cell types. Accumulating evidence demonstrates that BKCa is aberrantly expressed in many malignancies, involving in cancerous behaviors such as cell proliferation and migration. In this study, we investigated the functional role of BKCa in endometrial cancer HEC-1-B cells. Overexpression of BKCa by plasmid transfection enhanced endometrial cancer cell proliferation and migration. Conversely, silence of BKCa by lentivirus mediated RNAi system not only inhibited proliferation and migration but also impaired tumor growth in vivo. Patch clamp assay identified the BKCa currents in HEC-1-B cells, which was supported by the observation of channel activation or inhibition in response to the specific opener (NS1619) or blocker (IBTX) of BKCa. Moreover, NS1619 significantly increased cell proliferation and migration while IBTX exhibited the opposite effects. In summary, these data suggested an important role of BKCa in proliferation and migration of endometrial cancer HEC-1-B cells. Thus, BKCa may be established as a potential therapeutic target in endometrial cancer.

Molecular Rearrangements Underlying Function of Large Conductance Calcium- and Voltage-Regulated Potassium Channels

Molecular Rearrangements Underlying Function of Large Conductance Calcium- and Voltage-Regulated Potassium Channels

Effects of mechanical stretch on the functions of BK and L-type Ca2+ channels in vascular smooth muscle cells

It is well recognized that pathologically increased mechanical stretch plays a critical role in vascular remodeling during hypertension. However, how the stretch modulates the functions of ion channels of vascular smooth muscle cells (VSMCs) remains to be elucidated. Here, we demonstrated the effects of mechanical stretch on the activity of large conductance calcium, voltage-activated potassium (BK) and L-type Ca2+ channels. In comparison with 5% stretch (physiological), 15% stretch (pathological) upregulated the current density of L-type Ca2+ and BK channels as well as the frequency and amplitude of calcium oscillation in VSMCs. 15% stretch also increased the open probability and mean open time of the BK channel compared with 5% stretch. BK and L-type Ca2+ channels participated in the mechanical stretch-modulated calcium oscillation. Our results suggested that during hypertension, pathological stretch altered the activity of BK and L-type Ca2+ channels and manipulated the calcium oscillation of VSMCs.

Membrane depolarization activates BK channels through ROCK-mediated β1 subunit surface trafficking to limit vasoconstriction.

Membrane depolarization of smooth muscle cells (myocytes) in the small arteries that regulate regional organ blood flow leads to vasoconstriction. Membrane depolarization also activates large-conductance calcium (Ca2+)-activated potassium (BK) channels, which limits Ca2+ channel activity that promotes vasoconstriction, thus leading to vasodilation. We showed that in human and rat arterial myocytes, membrane depolarization rapidly increased the cell surface abundance of auxiliary BK β1 subunits but not that of the pore-forming BKα channels. Membrane depolarization stimulated voltage-dependent Ca2+ channels, leading to Ca2+ influx and the activation of Rho kinase (ROCK) 1 and 2. ROCK1/2-mediated activation of Rab11A promoted the delivery of β1 subunits to the plasma membrane by Rab11A-positive recycling endosomes. These additional β1 subunits associated with BKα channels already at the plasma membrane, leading to an increase in apparent Ca2+ sensitivity and activation of the channels in pressurized arterial myocytes and vasodilation. Thus, membrane depolarization activates BK channels through stimulation of ROCK- and Rab11A-dependent trafficking of β1 subunits to the surface of arterial myocytes.

Distinct domains of the β1-subunit cytosolic N terminus control surface expression and functional properties of large-conductance calcium-activated potassium (BK) channels

The properties and function of large-conductance calcium- and voltage-activated potassium (BK) channels are modified by the tissue-specific expression of regulatory β1-subunits. Although the short cytosolic N-terminal domain of the β1-subunit is important for controlling both BK channel trafficking and function, whether the same, or different, regions of the N terminus control these distinct processes remains unknown. Here we demonstrate that the first six N-terminal residues including Lys-3, Lys-4, and Leu-5 are critical for controlling functional regulation, but not trafficking, of BK channels. This membrane-distal region has features of an amphipathic helix that is predicted to control the orientation of the first transmembrane-spanning domain (TM1) of the β1-subunit. In contrast, a membrane-proximal leucine residue (Leu-17) controls trafficking without affecting functional coupling, an effect that is in part dependent on controlling efficient endoplasmic reticulum exit of the pore-forming α-subunit. Thus cell surface trafficking and functional coupling with BK channels are controlled by distinct domains of the β1-subunit N terminus.

Activation of N‐methyl‐D‐aspartate Receptor Inhibits Enkephalinergic Neurons in Mouse Spinal Superficial Dorsal Horn

Enkephalin (Enk)‐containing neurons are distributed at high concentration in the spinal cord superficial dorsal horn (SDH), where they play an important role in the transmission and modulation of nociceptive information. Recently, we performed physiological characterization of Enk‐containing neurons identified by green fluorescent protein fluorescence expressed under the control of the preproenkephalin gene promoter. We observed that Enk‐containing neurons in the SDH express the N‐methyl‐D‐aspartate receptor (NMDAR) 2B (NR2B) subunit and large conductance calcium activated potassium (BK) channels. In the present experiments, we investigated the involvement of NMDAR and BK channels in the regulation of the activity of Enk‐containing neurons in the SDH.The experiments were performed in 5‐ to 8‐week‐old male ICR mice. Under ketamine/xylazine anesthesia, the lumbar spinal cord was dissected, and transverse slices were prepared. Tight‐seal whole‐cell recordings were obtained from neurons in the SDH. L‐glutamate (Glu) was applied by pressure ejection through a glass pipette placed near the recorded neurons. After the electrophysiological experiments, the recorded neurons were sampled, and single cell real‐time RT‐PCR was performed to analyze the expression profile of genes of interest in single SDH neurons. In some experiments, the left sciatic nerve was partially ligated, and mechanical allodynia was assessed by von Frey test.Puff‐applied Glu evoked an inward current at a holding potential of −70 mV. Depolarizing the holding potential to 0 mV, outward current of long duration appeared subsequent to initial inward current. The NMDAR antagonist AP‐V abolished the outward current. The NR2B‐selective NMDAR antagonist ifenprodil reduced the outward current. The outward current was also abolished by the selective BK channels antagonist iberiotoxin. Single cell real‐time RT‐PCR analysis revealed that the neurons generating the outward current showed a significant tendency to express BK channels α‐subunit mRNA, which is consistent with the pharmacological results of a BK channels antagonist. Furthermore, single SDH neurons expressing the preproenkephalin mRNA also expressed the BK channel α‐subunit, NR1, and NR2B subunit mRNAs. The expression of NR2B increased significantly in sciatic nerve‐ligated mice.Our results suggest that Ca2+ influx through NMDAR may activate BK channels and thus inhibit Enk‐containing neurons in the SDH. NMDAR‐mediated inhibition of Enk‐containing neurons is speculated to be one of the possible cellular mechanisms of chronic pain induced by peripheral nerve injury.

Functional Coupling of BK Channels to NMDA Receptors in Hippocampal Dentate Gyrus

The large conductance calcium- and voltage-activated potassium channel (BK channels) is widely expressed in the central nervous system. The physiological roles of BK channels and the calcium sources of their activation in mammalian brain remain not well-understood. Our proteomic and biochemical analyses of BK channels and the calcium-permeable NMDA receptors and have found that they form protein complexes in whole brain and various brain regions, consisting of the obligatory BK channel alpha-subunit and GluN1 subunits and at least the regulatory GluN2A and GluN2B subunits. We examined the functional coupling of BK channels to NMDA receptors in mice hippocampal dentate gyrus. We observed that glutamate evoked outward currents of BK channels in dentate gyrus granule cell soma at potentials more positive than −20 mV which were sensitive to BK channel-specific blocker paxilline and abolished by NMDA receptor antagonist AP-5. We also found that blockade of BK channels by paxilline increased the amplitude of excitatory postsynaptic potential (EPSP) of granule cells evoked by stimulation in the perforant path-granule cell synapses, which was prevented by the presence of AP-5. We thus inferred that BK channels were activated by NMDA receptor-mediated Ca2+ influx in hippocampal dentate gyrus granule cells and the calcium-mediated functional coupling between these two types of channels plays a role in regulation of synaptic transmission.

BK Channel Gating-Ring Voltage Dependence Motions

The open probability of Large Conductance Voltage and Calcium dependent potassium (BK) channels is regulated allosterically by change in the transmembrane voltage and intracellular concentration of divalent ions (Ca2+ and Mg2+). BK channels are formed as tetramers of alpha subunits. The “divalent sensor” resides within the gating-ring, a large tetrameric structure formed by eight Regulator of Conductance of Potassium (RCK) domains, two from each subunit. Each RCK domain (RCK1 and RCK2) contains a high affinity- Ca2+binding site. Using Patch-clamp fluorometry, we have shown large changes in FRET signals within the gating-ring in response to Ca2+ and voltage. Here we investigate the origins of the voltage dependent motions. We tested manipulations that mostly change the BK channels’ relative probability of opening and in all cases the voltage dependence of the FRET signals remained within the voltage range of wild-type. However, mutations in the voltage sensor that are known to shift the charge displacement currents have a parallel shift of the gating ring's motions. These result suggest that the conformational changes of the gating ring are coupled to the voltage sensor, and could be relevant in the allosteric modulation of the BK channels.

Mechanosensitivity of Mitochondrial Potassium Channels

Mitochondria are known for their unique membrane architecture with the inner membrane folded into cristae. It is also known for long that mitochondria undergo drastic changes in volume. These volume changes depend on potassium fluxes. Influx via mitochondrial potassium channels (e.g. mitoBKCa or mitoKATP) results in swelling of mitochondria and unfolding of the cristae, which could be potentially translated into changes in the inner membrane tension. However, little is known about mechanobiology of mitochondria including existence of mechanosensitive channels. Mitochondrial large conductance calcium activated potassium channel BKCa (mitoBKCa) resides in the inner mitochondrial membrane (IMM) and plays an important role in cytoprotection. KCNMA1 gene encodes for both mitochondrial and plasmalemmal versions of BKCa channels, which share common features including large conductance, synergistic gating by voltage and Ca2+ ions. However, it was suggested that one of plasmalemmal splice variants of BKCa containing STREX exon was uniquely mechanosensitive. Here we show that mitoBKCa formed by another splice variant BKCa-DEC is also sensitive to stretch. We discuss potential physiological role of mechanosensitivity of mitoBKCa during mitochondria swelling.