Dynamic Changes In Intracellular Ca2 Research Articles

Evaluation of four KCNMA1 channelopathy variants on BK channel current under CaV1.2 activation

ABSTRACT Variants in KCNMA1, encoding the voltage- and calcium-activated K+ (BK) channel, are associated with human neurological disease. The effects of gain-of-function (GOF) and loss-of-function (LOF) variants have been predominantly studied on BK channel currents evoked under steady-state voltage and Ca2+ conditions. However, in their physiological context, BK channels exist in partnership with voltage-gated Ca2+ channels and respond to dynamic changes in intracellular Ca2+ (Ca2+ i). In this study, an L-type voltage-gated Ca2+ channel present in the brain, CaV1.2, was co-expressed with wild type and mutant BK channels containing GOF (D434G, N999S) and LOF (H444Q, D965V) patient-associated variants in HEK-293T cells. Whole-cell BK currents were recorded under CaV1.2 activation using buffering conditions that restrict Ca2+ i to nano- or micro-domains. Both conditions permitted wild type BK current activation in response to CaV1.2 Ca2+ influx, but differences in behavior between wild type and mutant BK channels were reduced compared to prior studies in clamped Ca2+ i. Only the N999S mutation produced an increase in BK current in both micro- and nano-domains using square voltage commands and was also detectable in BK current evoked by a neuronal action potential within a microdomain. These data corroborate the GOF effect of N999S on BK channel activity under dynamic voltage and Ca2+ stimuli, consistent with its pathogenicity in neurological disease. However, the patient-associated mutations D434G, H444Q, and D965V did not exhibit significant effects on BK current under CaV1.2-mediated Ca2+ influx, in contrast with prior steady-state protocols. These results demonstrate a differential potential for KCNMA1 variant pathogenicity compared under diverse voltage and Ca2+ conditions.

Preservation of endoplasmic reticulum (ER) Ca2+ stores by deletion of inositol-1,4,5-trisphosphate receptor type 1 promotes ER retrotranslocation, proteostasis, and protein outer segment localization in cyclic nucleotide-gated channel-deficient cone photoreceptors.

Endoplasmic reticulum (ER) Ca2+ homeostasis relies on an appropriate balance between efflux- and influx-channel activity responding to dynamic changes of intracellular Ca2+ levels. Dysregulation of this complex signaling network has been shown to contribute to neuronal and photoreceptor death in neuro- and retinal degenerative diseases, respectively. In mice with cone cyclic nucleotide-gated (CNG) channel deficiency, a model of achromatopsia/cone dystrophy, cones display early-onset ER stress-associated apoptosis and protein mislocalization. Cones in these mice also show reduced cytosolic Ca2+ level and subsequent elevation in the ER Ca2+ -efflux-channel activity, specifically the inositol-1,4,5-trisphosphate receptor type 1 (IP3 R1), and deletion of IP3 R1 results in preservation of cones. This work investigated how preservation of ER Ca2+ stores leads to cone protection. We examined the effects of cone specific deletion of IP3 R1 on ER stress responses/cone death, protein localization, and ER proteostasis/ER-associated degradation. We demonstrated that deletion of IP3 R1 improves trafficking of cone-specific proteins M-/S-opsin and phosphodiesterase 6C to cone outer segments and reduces localization to cone inner segments. Consistent with the improved protein localization, deletion of IP3 R1 results in increased ER retrotranslocation protein expression, reduced proteasome subunit expression, reduced ER stress/cone death, and reduced retinal remodeling. We also observed the enhanced ER retrotranslocation in mice that have been treated with a chemical chaperone, supporting the connection between improved ER retrotranslocation/proteostasis and alleviation of ER stress. Findings from this work demonstrate the importance of ER Ca2+ stores in ER proteostasis and protein trafficking/localization in photoreceptors, strengthen the link between dysregulation of ER Ca2+ homeostasis and ER stress/cone degeneration, and support an involvement of improved ER proteostasis in ER Ca2+ preservation-induced cone protection; thereby identifying IP3 R1 as a critical mediator of ER stress and protein mislocalization and as a potential target to preserve cones in CNG channel deficiency.

Dynamic changes of intracellular Ca2+ content of rats brain after traumatic brain injury and its correlation with the concentration of calcitonin gene‐related peptide

ObjectiveTo investigate the dynamic changes of intracellular Ca2+ content of rats brain after traumatic brain injury (TBI) and its correlation with the concentration of calcitonin gene‐related peptide (CGRP)MethodsThe rats model with TBI was prepare by Feeney’s methods. 144 male Wistar rats were randomly divided into Sham operation group(Sham group, 36 rats) and traumatic brain injury group (TBI group, 108 rats ). Above two groups subdivided into six groups by 2h,6h,12h,24h, 48h and 72h after TBI( 6 rats in each group. The calcium content in the rats brain cells were detected by laser scanning confocal microscope, and the CGRP concentration in plasma of rats were got by enzyme‐linked immunosorbent assay.ResultsThere were significant differences of calcium fluorescence intensity in the rats brain cells between the Sham group and the TBI group (P<0.05). In the meantime there were also significant differences of the CGRP concentration in plasma of rats between the Sham group and the TBI group (P<0.05). After the injury, calcium fluorescence intensity in the rats brain cells began to rise within 2 hours, reached its peak in 24 hours, and decreased in 48 hours (P<0.05). In the TBI group, the CGRP concentration in plasma of rats increased at 2h, then decreased at 24h,48h, and increased slowly at 72h after TBI (P<0.05).ConclusionThere was a negative correlation between CGRP concentration in rats plasma and the intracellular calcium content of rats brain. Calcium overload played an important role in the brain secondary damage after TBI.Support or Funding Information• This study was supported by the National Natural Science Foundation Youth Fund (30600637), China Postdoctral Science Foundation Grant (2019T120195), China Postdoctral Science Foundation Grant (2014M561207), the Shanxi Province Basic Research Program ( 2010021034‐4, 201601D011119), the Shanxi Scholarship Council of China (2011‐096 and Key Projects No.4), Department of Human Resources and Social Security of Shanxi province fund grant, The science and technology research projects of health department of Shanxi Province(2016D011119), The Teaching Reform Program of Shanxi Higher Education, The Education Reform Project of Postgraduate Students in Shanxi Province. The Project of “131” Leading Talents of Shanxi Higher Education, The Education Reform Project of Shanxi Medical University, the fund grant of the eleventh college of shanxi medical university(Lvliang Hospital).

The CBL-CIPK Calcium Signaling Network: Unified Paradigm from 20 Years of Discoveries.

Calcium (Ca2+) serves as an essential nutrient as well as a signaling agent in all eukaryotes. In plants, calcineurin B-like proteins (CBLs) are a unique group of Ca2+ sensors that decode Ca2+ signals by activating a family of plant-specific protein kinases known as CBL-interacting protein kinases (CIPKs). Interactions between CBLs and CIPKs constitute a signaling network that enables information integration and physiological coordination in response to a variety of extracellular cues such as nutrient deprivation and abiotic stresses. Studies in the past two decades have established a unified paradigm that illustrates the functions of CBL-CIPK complexes in controlling membrane transport through targeting transporters and channels in the plasma membrane and tonoplast.

Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on micropillar substrates

In this study, poly-L-lactic acid micropillar substrates were fabricated to evaluate the influence of topographic substrates on cell morphological and functional characteristics, such as spreading area, voltage-gated calcium channels (VGCCs) and membrane potential. The proliferation, spreading area, perimeter and circularity of SH-SY5Y cells interfaced with different substrates were first investigated. In addition, the cytoskeleton and focal adhesion of a cell as important manifestations of cell morphology were analyzed by immunofluorescence. VGCC responsiveness was evaluated by measuring the dynamic changes in intracellular Ca2+ evoked by 50 mM extracellular K+. To determine study whether the differences in VGCC responsiveness were caused by the differences in VGCC gene expression, the expression of N/L- type VGCCs was determined by qPCR and fluorescence staining. Notably, improved measurement of the membrane potential with potentiometric fluorescent dye TMRM was applied to determine the membrane potential of SH-SY5Y cells. Results indicated that the SH-SY5Y cells were deformed significantly to adapt to the substrates; however, no distinct effect on the proliferative ability of SH-SY5Y cells was observed. The micropillar substrates markedly influenced VGCC responsiveness, which correlated strongly with cell spreading but not with VGCC expression. The resting membrane potential of SH-SY5Y cells cultured on different substrates also changed, but no effect on responsiveness of VGCC was observed. These results suggest that the effect of the micropillar substrates on cell VGCC responsiveness may be attributed to changes in the functionality of the ion channel itself. Thus, topographic substrates can be used to engineer cell functionality in cell-based drug screening.

Astrocyte dysfunction in Alzheimer disease.

Astrocytes are glial cells that are distributed throughout the central nervous system in an arrangement optimal for chemical and physical interaction with neuronal synapses and brain blood supply vessels. Neurotransmission modulates astrocytic excitability by activating an array of cell surface receptors and transporter proteins, resulting in dynamic changes in intracellular Ca2+ or Na+ . Ionic and electrogenic astrocytic changes, in turn, drive vital cell nonautonomous effects supporting brain function, including regulation of synaptic activity, neuronal metabolism, and regional blood supply. Alzheimer disease (AD) is associated with aberrant oligomeric amyloid β generation, which leads to extensive proliferation of astrocytes with a reactive phenotype and abnormal regulation of these processes. Astrocytic morphology, Ca2+ responses, extracellular K+ removal, glutamate transport, amyloid clearance, and energy metabolism are all affected in AD, resulting in a deleterious set of effects that includes glutamate excitotoxicity, impaired synaptic plasticity, reduced carbon delivery to neurons for oxidative phosphorylation, and dysregulated linkages between neuronal energy demand and regional blood supply. This review summarizes how astrocytes are affected in AD and describes how these changes are likely to influence brain function. © 2017 Wiley Periodicals, Inc.

Smooth Muscle Cell Ca2+: Think Locally, Act Globally

Dynamic changes in intracellular Ca²⁺ levels in vascular smooth muscle cells are critically important for cardiovascular regulation. This Special Topic Issue highlights a series of expert opinion articles focused on this important subject. After a brief overview, novel discoveries surrounding smooth muscle cell Ca²⁺ influx via L-type and T-type channels are reviewed. Current work revealing the functional importance of dynamic Ca²⁺ signaling in the control of the parenchymal microvasculature and the emerging role of mitochondrial Ca²⁺ signaling and store-operated Ca²⁺ entry in smooth muscle cells is discussed. Finally, recent data describing a new target of localized Ca²⁺ signaling in arterial myocytes that is responsible for membrane depolarization is reviewed. Authors were encouraged to write in an opinionated and provocative manner with the hope of stimulating discussion in this area of research.

Intercellular Ca2+ wave propagation in human retinal pigment epithelium cells induced by mechanical stimulation

Ca2+ signaling is vitally important in cellular physiological processes and various drugs also affect Ca2+ signaling. Thus, knowledge of Ca2+ dynamics is important toward understanding cell biology, as well as the development of drug-testing assays. ARPE-19 cells are widely used for modeling human retinal pigment epithelium functions and drug-testing, but intercellular communication has not been assessed in these cells. In this study, we investigated intercellular Ca2+ communication induced by mechanical stimulation in ARPE-19 cells. An intercellular Ca2+ wave was induced in ARPE-19 monolayer by point mechanical stimulation of a single cell. Dynamic changes of intracellular Ca2+ concentration ([Ca2+]i) in the monolayer were tracked with fluorescence microscopy imaging using Ca2+-sensitive fluorescent dye fura-2 in presence and absence of extracellular Ca2+, after depletion of intracellular Ca2+ stores with thapsigargin, and after application of gap junction blocker α-glycyrrhetinic acid and P2-receptor blocker suramin. Normalized fluorescence values, reflecting amplitude of [Ca2+]i increase, and percentage of responsive cells were calculated to quantitatively characterize Ca2+ wave propagation. Mechanical stimulation of a single cell within a confluent monolayer of ARPE-19 cells initiated an increase in [Ca2+]i, which propagated to neighboring cells in a wave-like manner. Ca2+ wave propagated to up to 14 cell tiers in control conditions. The absence of extracellular Ca2+ reduced [Ca2+]i increase in the cells close to the site of mechanical stimulation, whereas the depletion of intracellular Ca2+ stores with thapsigargin blocked the wave spreading to distant cells. The gap junction blocker α-glycyrrhetinic acid reduced [Ca2+]i increase in the cell tiers close to the site of mechanical stimulation, indicating involvement of gap junctions in Ca2+ wave propagation. The P2-receptor blocker suramin reduced the percentage of responsive cells participating in Ca2+ wave spreading beyond the fourth cell tier, showing the necessity of P2-receptors for Ca2+ wave propagation. In disconnected, i.e., subconfluent, ARPE-19 cell clusters Ca2+ wave spreading was considerably less efficient compared to that in confluent ARPE-19 monolayer at the same distances. ARPE-19 cells showed repeatable and robust Ca2+ dynamics after mechanical stimulus. The ARPE-19 cells exhibited two different mechanisms of Ca2+ wave propagation dependent on the cell location: in the cells close to the site of mechanical stimulation the Ca2+ wave propagated mainly through gap junctions and required Ca2+ from both intracellular Ca2+ stores and extracellular media, while farther away the propagation was more dependent on the purinergic receptors and did not require extracellular Ca2+. The proposed method could provide a tool to assess the drug-induced changes in intercellular communication in in vitro assays in human retinal pigment epithelial cells.

Interleukin-6 Reduces NMDA-Induced Ca2+ Overload via Prevention of Ca2+ Release From Intracellular Store

ABSTRACTA mechanism for neuroprotection of interleukin-6 (IL-6) via reduction of intracellular Ca2+ overload induced by N-methyl-D-aspartate (NMDA) was explored. Cerebellar granule neurons (CGNs) from postnatal 8-day infant rats were chronically exposed to IL-6 for 8 days. Confocal laser scanning microscope was used to measure dynamic changes of intracellular Ca2+ fluorescence intensity. NMDA triggered an acute and sustaining enhancement of intracellular Ca2+ fluorescence intensity in the cultured CGNs, whereas NMDA stimulation of the neurons that had been exposed to IL-6 reduced the intracellular Ca2+ fluorescence intensity relative to that of non-IL-6-pretreated neurons. Ethylene glycol bis(aminoethylether) tetraacetate (EGTA), a chelator of extracellular Ca2+, decreased the intracellular Ca2+ overload triggered by NMDA. The component of intracellular Ca2+ overload after EGTA treatment was prevented by IL-6 chronic exposure. Cotreatment with dantrolene sodium (DAN) and 2-aminoethoxydiphenylborate (2-APB), blockers of ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP3R), respectively, also decreased the intracellular Ca2+ overload triggered by NMDA. However, the component of intracellular Ca2+ overload after DAN and 2-APB treatment was only slightly but not significantly diminished by IL-6. These results suggest that IL-6 has a neuroprotective effect against NMDA-induced intracellular Ca2+ overload, and that the effect is implemented primarily via a suppression of Ca2+ release from the intracellular Ca2+ store.

Role of Membrane Potential in Calcium Signaling During Rhythmic Bursting in Tritonia Swim Interneurons

Rhythmic bursting in neurons is accompanied by dynamic changes in intracellular Ca(2+) concentration. These Ca(2+) signals may be caused by membrane potential changes during bursting and/or by synaptic inputs. We determined that membrane potential is responsible for most, if not all, of the cytoplasmic Ca(2+) signal recorded during rhythmic bursting in two neurons of the escape swim central pattern generator (CPG) of the mollusk, Tritonia diomedea: ventral swim interneuron B (VSI) and cerebral neuron 2 (C2). Ca(2+) signals were imaged with a confocal laser scanning microscope while the membrane potential was recorded at the soma. During the swim motor pattern (SMP), Ca(2+) signals in both neurons transiently increased during each burst of action potentials with a more rapid decay in secondary than in primary neurites. VSI and C2 were then voltage-clamped at the soma, and each neuron's own membrane potential waveform recorded during the SMP was played back as the voltage command. In all regions of VSI, this completely reproduced the amplitude and time course of Ca(2+) signals observed during the SMP, but in C2, the amplitude was lower in the playback experiments than during the SMP, possibly due to space clamp problems. Therefore in VSI, the cytoplasmic Ca(2+) signal during the SMP can be accounted for by its membrane potential excursions, whereas in C2 the membrane potential excursions can account for most of the SMP Ca(2+) signal.

Inhibitory effects of extracellular Mg2+ on intracellular Ca2+ dynamic changes and thapsigargin-induced apoptosis in human cancer MCF7 cells.

The effects of extracellular Mg2+ on both dynamic changes of [Ca2+]i and apoptosis rate were analysed. The consequences of spatial and temporal dynamic changes of intracellular Ca2+ on apoptosis, in thapsigargin- and the calcium-ionophore 4BrA23187-treated MCF7 cells were first determined. Both 4BrA23187 and thapsigargin induced an instant increase of intracellular Ca2+ concentrations ([Ca2+]i) which remained quite elevated (> 150 nM) and lasted for several hours. [Ca2+]i increases were equivalent in the cytosol and the nucleus. The treatments that induced apoptosis in MCF7 cells were systematically associated with high and sustained [Ca2+]i (150 nM) for several hours. The initial [Ca2+]i increase was not determinant in the events triggering apoptosis. Thapsigargin-mediated apoptosis and [Ca2+]i rise were abrogated when cells were pretreated with the calcium chelator BAPTA. The role of the extracellular Mg2+ concentration has been studied in thapsigargin treated cells. High (10 mM) extracellular Mg2+, caused an increase in basal [Mg2+]i from 0.8+/-0.3 to 1.6+/-0.5 mM. As compared to 1.4 mM extracellular Mg2+, 1 microM thapsigargin induces, in 10 mM Mg2+, a reduced percentage from 22 to 11% of fragmented nuclei, a lower sustained [Ca2+]i and a lower Ca2+ influx through the plasma membrane. In conclusion, the cell death induced by thapsigargin was dependent on high and sustained [Ca2+]i which was inhibited by high extracellular and intracellular Mg2+.

Extracellular Mg2+Inhibits both Histamine-Stimulated Ca2+-Signaling and Exocytosis in Human Tracheal Secretory Gland Cells

The effects of extracellular Mg2+concentration have been investigated on the histamine-stimulated exocytotic process of human tracheal secretory gland (HTG) cells. The exocytosis of secretory granules (SG) was observed concomitantly with dynamic changes of intracellular Ca2+([Ca2+]i) and Mg2+concentrations ([Mg2+]i). The rate of SG exocytosis was appraised by the decrease of quinacrine fluorescence emission. Dynamic changes of [Mg2+]iand [Ca2+]iin HTG cells were determined by the combined use of UV-microspectrofluorometry with Mag-Indo-1 and Indo-1 probes, respectively. High Mg2+medium significantly inhibited the histamine-stimulated secretion. The influence of the extracellular and intracellular Mg2+concentrations on [Ca2+]iwas analyzed. Basal [Mg2+]iincreased from 0.8 mM in a Mg2+-free medium to 1.7 mM in 10 mM Mg2+medium. Histamine induced a [Mg2+]iincrease which is dependent on extracellular Mg2+concentration. The histamine stimulated [Ca2+]irise was reduced in the presence of elevated Mg2+extracellular medium and inhibitory effects of extracellular Mg2+were concomitant with changes in [Mg2+]i. Our data suggest that the inhibition by extracellular Mg2+of stimulated exocytosis is dependent on both the increase of [Mg2+]iand the inhibition of cytosolic Ca2+influx.

Mechanisms of vasopressin-induced increase in intracellular Na+ in vascular smooth muscle cells.

The present study was undertaken to examine the interrelationship between the arginine vasopressin (AVP)-induced dynamic changes in intracellular Ca2+ and Na+ concentrations in cultured rat aortic vascular smooth muscle cells (VSMC) by the direct measurements of intracellular Na+ concentration [( Na+]i), cytosolic free calcium [( Ca2+]i), and intracellular pH (pHi) using fluorescence dyes. AVP increased [Ca2+]i and [Na+]i in a dose-dependent manner; the rise in [Ca2+]i preceded the rise in [Na+]i. Pretreatment with the V1 antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid) 2-(O-methyl)-tyrosine] AVP [( d(CH2)5Tyr(Me)]AVP) completely blocked the effects of AVP on [Ca2+]i and [Na+]i. The V2 agonist 1-desamino-8-D-AVP, DDAVP, did not affect basal [Na+]i or the AVP-induced increase in [Na+]i. Also, Ca(2+)-free solution completely blocked the AVP-induced increase in [Na+]i. Moreover, Ca(2+)-free solution decreased the AVP-induced intracellular acidification and blunted the later AVP-induced intracellular alkalinization. These results therefore indicate that after binding to the V1 receptor AVP increases [Na+]i mediated by the activation of Na(+)-Ca2+ and Na(+)-H+ exchanges in VSMC. All of these cellular events are completely dependent on an increase in cellular Ca2+ uptake produced by AVP.