Identification And Regulatory Effects Of Nuclear Large Conductance Calcium Activated Potassium Channels On Nuclear Calcium Signaling

As an important second messenger in the cell,calcium participates in a variety of physiological and pathological processes including muscle contraction, neurotransmitter release,gene expression,cell proliferation and apoptosis.The changes of intracellular calcium concentration are not a synchronizing process both temporally and spatially.In different stimulations and cell activities,calcium concentration of different organelles and sub-cellular compartments are not same and participate in different cellular events.For example,in synapses of nervous system, neural impulses stimulate the elevation of presynaptic calcium concentration,which could induce the neurotransmitter release,thus accomplish the neural transmission in synapses.As the energy factory in the cell,mitochondria have their own calcium transport system.Changes of mitochondrial calcium concentration will influence ATP production and permeability of mitochondrial membrane,thus mediate cell apoptosis.As one of the most important organelles in the cell,nucleus is the participant and location of many cellular events.The alteration of nuclear calcium concentration could influence gene expression,nuclear protein and RNA transport,DNA and nuclear protein degradation,cell division and other cellular events.Moreover,it was found recently that nuclear calcium itself is essential to trigger cell proliferation and synaptic activity induced gene expression.Therefore,discovering nuclear calcium production and regulation mechanisms is important for elucidating cell functions.Nucleus is surrounded by nuclear envelope,which consists of inner membrane and outer membrane.Between the two membranes is the perinuclear space,which is continuous with the lumen of endoplasmic reticulum（ER）.The inner and outer membranes join periodically at the nuclear pore complex（NPC）.It has long been assumed that the NPC forms a large channel for the free diffusion of ions and macromolecules.Calcium could diffuse into and out of nucleoplasm through NPC, and the alteration of nuclear calcium concentration is dependent on cytoplasmic calcium concentration.But the it was found that NPCs are not always in a open state by using patch clamp and atomic force microscope.The NPC could be blocked by the central plug,thus the permeability of NPC is very low.It was found in recent studies that perinuclear space could serve as a calcium store and be a nuclear calcium resource.Calcium pumps（calcium ATPases） located in the outer membrane pump the calcium into the perinuclear space and IP3receptors and ryanodine receptors located in the inner membrane mediate the calcium release to nucleoplasm.But the regulatory mechanism of this process is to be elucidated.This study found that large conductance calcium activated potassium channels (BK_Ca channels) express in the nuclear envelope and regulate calcium release from perinuclear space to nucleoplasm.1.The expression of BK_Ca channels in the nuclear envelope of primary cultured hippocampal neurons.In order to confirm the expression of BK_Ca channels in the nuclear envelope,we isolated and purified the hippocampal neurons nuclei.The expression of BK_Ca channels in the nuclear envelope was confirmed by BK_Ca channels C terminus specific antibodies and fluorescence labeled secondary antibodies.Since we did not use Triton-X100 during the experiment,the nuclear envelope is impermeable to the antibodies,and antibodies could not enter the perinuclear space,therefore the detected expression of BK_Ca channels suggested C terminus of BK_Ca channels faces the cytoplasmic side. Overexpression of BK_Ca channels and green fluorescence protein（GFP） fusion protein in neurons and U87 cells illustrated the co-localization of BK_Ca channels and lamin B,a nuclear envelope marker.C terminus of BK_Ca channels and GFP fusion protein could not localized in nuclear envelope,which suggested that the localization of BK_Ca channels in the nuclear envelope depends on the transmembrane domains.The localization of BK_Ca channels in nuclear envelope was further confirmed by western blot experiments of isolated nuclei.The expression of BK_Ca channels could not be detected in denuded nuclei.Furthermore,the expression of BK_Ca channels in nuclei was detected in nuclear fraction purified by a commercial kit.The expression of BK_Ca channels in nuclear fraction increase when U87 cells were transfected with BK_Ca channels plasmids compare with the control plasmids,and miRNA against BK_Ca channels could attenuate the BK_Ca channels expression,which further confirm the expression of BK_Ca channels in nuclei.We alse recorded a potassium channel activity with a similar single channel conductance with BK_Ca channels,which could be inhibited by paxilline.2.Nuclear BK_Ca channels could regulate nuclear envelope potential and nuclear calcium signaling.DiOC₆（3） is a voltage sensitive dye,and it will located in the nuclear envelope by its lipophillic property.The fluorescence intensity of DiOC₆（3） reflects the nuclear envelope potential（ΔΨn）.BK_Ca channels specific antagonist paxilline could increase the fluorescence intensity of DiOC₆（3）,which suggested that paxilline could make the nuclear envelope potential more negative and hyperpolarized.While the NS1619,a BK_Ca channels specific agonist,could decrease the fluorescence intensity of DiOC₆（3）, which suggested that NS1619 could depolarize the nuclear envelope potential.Calcium indicator Fluo4-AM,a cell permeable dye,located mainly in the perinuclear space,whose fluorescence intensity reflects the calcium concentration in perinuclear space.On the contrary,Calcium indicator Fluo4-dextran（10,000MW）,a cell impermeable dye,mainly located in nucleoplasm,whose fluorescence intensity reflects the calcium concentration in nucleoplasm.When applied to isolated nuclei, BK_Ca channels specific antagonist paxilline could decrease the fluorescence intensity of Fluo4-AM but increase that of Fluo4-dextran,which suggested paxilline could induce the calcium release from perinuclear space to nucleoplasm.This process could be blocked by ruthenium red,a ryanodine receptor antagonist,and thapsigargin,a calcium pump inhibitor,but could not be blocked by IP3 receptor heparin,which suggested that the effects of paxilline on nuclear calcium release depended on the activity of ryanodine receptor.IbTx,another BK_Ca channels specific antagonist,is a peptide and impermeable to cell membrane.Ibtx blocks pore of BK_Ca channels in extracellular side.When applied to isolated nuclei,IbTx did not induce calcium concentration alteration.Together with the results in immunocytochemistry experiment,these results suggested that the orientation of BK_Ca channels in the nuclear is C terminus facing the cytosol and the pore which could be blocked by IbTx facing the perinuclear space.3.Nuclear BK_Ca channels could regulate depolarization induced[Ca²⁺]_cyto and [Ca²⁺]_nu coupling.Depolarization could induce elevation of cytosolic calcium concentration ([Ca²⁺]_cyto),followed by the elevation of nuclear calcium concentration([Ca²⁺]_nu). [Ca²⁺]_cyto could encode the synaptic activity,while[Ca²⁺]_nu is an important step for gene expression.Thus the[Ca²⁺]_cyto and[Ca²⁺]_nu coupling is the bridge of synaptic activity and gene expression.Synaptic activity to alteration of[Ca²⁺]_cyto,to alteration of[Ca²⁺]_nu,to transcription factor activation,neurons could convert the long term alteration of synaptic activity to gene expression by this signaling pathway.So discovering the[Ca²⁺]_cyto and[Ca²⁺]_nu coupling is the key to fully understand the relationship between synaptic activity and gene expression.Our results showed that BK_Ca channels specific antagonist paxilline could increase[Ca²⁺]_cyto and[Ca²⁺]_nu of culture hippocampal neurons;after paxilline pretreatment,depolarized stimulation （KCl） increase the slope of[Ca²⁺]_nu elevation rather than its amplitude.These results suggested that paxilline could participate in genetic encoding of synaptic activity by regulating nuclear calcium concentration.4.Nuclear BK_Ca channels regulate CREB phosphorylation.cAMP Response Element Binding Protein（CREB） is an important downstream signal molecule of calcium.CREB is phosphorylated when calcium concentration increases.The phosphorylated CREB（p-CREB） could bind to its promoter and activate downstream gene expression.Since BK_Ca channels could regulate nuclear calcium concentration,we further detected whether BK_Ca channels could regulate CREB phosphorylation by regulating nuclear calcium concentration.We used BK_Ca channels specific antagonist paxilline or BK_Ca channels specific agonist NS1619 to incubate isolated nuclei of hippocampal neurons.Western blot results demonstrated that paxilline could increase CREB phosphorylation,using low calcium solution and high calcium solution as controls.We got the similar results in immunocytochemisty experiments.These results suggested that paxilline could increase CREB phosphorylation by promoting calcium release from perinuclear space to nucleoplasm.To summarize,we found BK_Ca channels expressed in nuclear envelope and participate in the regulation of nuclear signaling.As an important potassium channel in the cell,BK_Ca channels could regulate intracellular calcium concentration in two positions.The activation of BK_Ca channels in the cell membrane makes the cell membrane hyperpolarize and decrease the open probability of voltage sensitive calcium channels,thus attenuate the calcium influx induced by synaptic activity.The BK_Ca channels in the nuclear envelope could regulate nuclear calcium concentration by regulating the nuclear envelope potential, thus influence the transcription factor activation and synaptic activity induced gene expression.The discovering and exploring of nuclear BK_Ca channels functions are pivotal to fully understand the calcium signaling pathways,regulation mechanisms and cell functions.It also helps to elucidate the mechanisms of synaptic activity related events,such as learning and memory,and nervous system diseases.