共查询到20条相似文献,搜索用时 15 毫秒
1.
Giacomello M De Mario A Lopreiato R Primerano S Campeol M Brini M Carafoli E 《Cell calcium》2011,(6):569-576
The inner ear converts sound waves into hearing signals through the mechanoelectrical transduction (MET) process. Deflection of the stereocilia bundle of hair cells causes the opening of channels that allow the entry of endolymph K+ and Ca2+. Ca2+ that enters is crucial to the hearing process and is exported to the endolymph by the plasma membrane Ca2+ pump (isoform PMCA2w/a): disturbances of the balance between Ca2+ penetration and ejection, e.g. by pump mutations, generate deafness. Hearing loss caused by PMCA defects is frequently exacerbated by mutations in cadherin 23, a single pass stereociliar Ca2+ binding protein that forms the tip links which permit the deflection of the stereocilia bundle and thus the opening of the MET channels. The PMCA2w/a pump ejects Ca2+ to the endolymph even in the absence of the natural activator calmodulin. This satisfies the special Ca2+ homeostasis requirements of the stereocilia/endolymph system. Here we have analyzed a mice and a human previously described pump mutant. The human mutant only exacerbated the deafness produced by a cadherin 23 mutation. The murine mutant overexpressed in model cells displayed an evident defect both in the basal activity of the pump and in the long range ejection of Ca2+, the human mutant instead failed to impair the Ca2+ ejection by the pump. 相似文献
2.
3.
Transient transfection of hair cells has proven challenging. Here we describe modifications to the Bio-Rad Helios Gene Gun that, along with an optimized protocol, improve transfection of bullfrog, chick, and mouse hair cells. The increased penetrating power afforded by our method allowed us to transfect mouse hair cells from the basal side, through the basilar membrane; this configuration protects hair bundles from damage during the procedure. We characterized the efficiency of transfection of mouse hair cells with fluorescently-tagged actin fusion protein using both the optimized procedure and a published procedure; while the efficiency of the two methods was similar, the morphology of transfected hair cells was improved with the new procedure. In addition, using the improved method, we were able to transfect hair cells in the bullfrog sacculus and chick cochlea for the first time. We used fluorescent-protein fusions of harmonin b (USH1C) and PMCA2 (ATP2B2; plasma-membrane Ca2+-ATPase isoform 2) to examine protein distribution in hair cells. While PMCA2-EGFP localization was similar to endogenous PMCA2 detected with antibodies, high levels of harmonin-EGFP were found at stereocilia tapers in bullfrog and chick, but not mouse; by contrast, harmonin-EGFP was concentrated in stereocilia tips in mouse hair cells. 相似文献
4.
Dakshnamurthy Selvakumar Marian J. Drescher Dennis G. Drescher 《The Journal of biological chemistry》2013,288(10):7215-7229
5.
Neeliyath A. Ramakrishnan Marian J. Drescher Roberto L. Barretto Kirk W. Beisel James S. Hatfield Dennis G. Drescher 《The Journal of biological chemistry》2009,284(5):3227-3238
The cytoplasmic amino terminus of HCN1, the primary full-length HCN isoform
expressed in trout saccular hair cells, was found by yeast two-hybrid
protocols to bind the cytoplasmic carboxyl-terminal domain of a protocadherin
15a-like protein. HCN1 was immunolocalized to discrete sites on saccular hair
cell stereocilia, consistent with gradated distribution expected for tip link
sites of protocadherin 15a. HCN1 message was also detected in cDNA libraries
of rat cochlear inner and outer hair cells, and HCN1 protein was
immunolocalized to cochlear hair cell stereocilia. As predicted by the trout
hair cell model, the amino terminus of rat organ of Corti HCN1 was found by
yeast two-hybrid analysis to bind the carboxyl terminus of protocadherin 15
CD3, a tip link protein implicated in mechanosensory transduction. Specific
binding between HCN1 and protocadherin 15 CD3 was confirmed with pull-down
assays and surface plasmon resonance analysis, both predicting dependence on
Ca2+. In the presence of calcium chelators, binding between HCN1
and protocadherin 15 CD3 was characterized by a KD = 2.39
× 10-7 m. Ca2+ at 26.5-68.0
μm promoted binding, with KD = 5.26 ×
10-8 m (at 61 μm Ca2+). Binding
by deletion mutants of protocadherin 15 CD3 pointed to amino acids 158-179
(GenBank™ accession number ), with homology to the comparable
region in trout hair cell protocadherin 15a-like protein, as necessary for
binding to HCN1. Amino terminus binding of HCN1 to HCN1, hypothesized to
underlie HCN1 channel formation, was also found to be
Ca2+-dependent, although the binding was skewed toward a lower
effective maximum [Ca2+] than for the HCN1 interaction with
protocadherin 15 CD3. Competition may therefore exist in vivo between
the two binding sites for HCN1, with binding of HCN1 to protocadherin 15 CD3
favored between 26.5 and 68 μ XP_238200m Ca2+. Taken together,
the evidence supports a role for HCN1 in mechanosensory transduction of inner
ear hair cells.HCN12 is the
primary full-length HCN isoform underlying Ih
(hyperpolarization-activated, cyclic nucleotide-gated, nonselective cation
channel current) in a model hair cell preparation from the trout sacccule
(1). cAMP-gated
Ih, possibly in addition to the
mechanosensory-transduction current, sets the membrane potential for a
subpopulation of saccular hair cells
(2,
3). The membrane potential in
the saccular hair cell subpopulation is sufficiently depolarized to activate
voltage-gated calcium channels, permitting influx of calcium and secretion of
hair cell transmitter (2).
Given that saccular hair cells expressing IK1 in addition
to Ih are more hyperpolarized, not supporting activation
of the voltage-gated calcium channels, we predicted that spontaneous release
of transmitter from the subpopulation of hair cells would constitute hair
cell-generated spontaneous activity for the saccule
(1). However, little has been
previously reported on the morphological localization of the HCN1 isoform in
hair cells or possible links to structural proteins that mechanistically would
localize HCN1 in hair cells (for preliminary report, see Ref.
4). In general, little is known
about protein-protein interactions for the HCN isoforms that would modulate
Ih and/or the associated instantaneous current
(5).Protocadherin 15 is a proposed tip link protein involved in connecting
shorter stereocilia to adjacent taller stereocilia in the stereociliary array
of inner ear hair cells, facilitating the opening of the mechanosensory
transduction channel in response to auditory and vestibular stimuli. The
active tip link protein in Danio rerio is protocadherin 15a
(6), characterized by splice
variants in its carboxyl terminus. In the mammal, protocadherin 15 CD3 is
hypothesized to be a tip link protein at insertion sites in the tips of the
shorter stereocilia of the stereociliary array
(7,
8). 相似文献
6.
Laura K. Gunther Joseph A. Cirilo Jr. Rohini Desetty Christopher M. Yengo 《Molecular biology of the cell》2022,33(1)
Class III myosins are actin-based motors proposed to transport cargo to the distal tips of stereocilia in the inner ear hair cells and/or to participate in stereocilia length regulation, which is especially important during development. Mutations in the MYO3A gene are associated with delayed onset deafness. A previous study demonstrated that L697W, a dominant deafness mutation, disrupts MYO3A ATPase and motor properties but does not impair its ability to localize to the tips of actin protrusions. In the current study, we characterized the transient kinetic mechanism of the L697W motor ATPase cycle. Our kinetic analysis demonstrates that the mutation slows the ADP release and ATP hydrolysis steps, which results in a slight reduction in the duty ratio and slows detachment kinetics. Fluorescence recovery after photobleaching (FRAP) of filopodia tip localized L697W and WT MYO3A in COS-7 cells revealed that the mutant does not alter turnover or average intensity at the actin protrusion tips. We demonstrate that the mutation slows filopodia extension velocity in COS-7 cells which correlates with its twofold slower in vitro actin gliding velocity. Overall, this work allowed us to propose a model for how the motor properties of MYO3A are crucial for facilitating actin protrusion length regulation. 相似文献
7.
The cell membranes in the hair bundle of an auditory hair cell confront a difficult task as the bundle oscillates in response to sound: for efficient mechanotransduction, all the component stereocilia of the hair bundle must move essentially in unison, shearing at their tips yet maintaining contact without membrane fusion. One mechanism by which this cohesion might occur is counterion-mediated attachment between glycan components of apposed stereociliary membranes. Using capillary electrophoresis, we showed that the stereociliary glycocalyx acts as a negatively charged polymer brush. We found by force-sensing photomicrometry that the stereocilia formed elastic connections with one another to various degrees depending on the surrounding ionic environment and the presence of N-linked sugars. Mg2+ was a more potent mediator of attachment than was Ca2+. The forces between stereocilia produced chaotic stick-slip behavior. These results indicate that counterion-mediated interactions in the glycocalyx contribute to the stereociliary coherence that is essential for hearing. 相似文献
8.
The presence and localization of voltage-gated Ca2+ channels of L-type were investigated in intestinal cells of the Atlantic cod. Enterocytes were loaded with the fluorescent
Ca2+ probe, fure-2/AM and changes in intracellular Ca2+ concentrations ([Ca2+]
i
) were measured, in cell suspensions, in the presence of high potassium levels (100 mm), BAY K-8644 (5 μm), nifedipine (5 μm) or ω-conotoxin (1 μm). L-type Ca2+ channels were visualized on intestinal sections using the fluorescent dihydropyridine (-)-STBodipy.
Depolarization of the plasma membrane produced a rapid (within 5 sec) and transient (at basal levels after 21 sec) increase
in [Ca2+]
i
. BAY K-8644 increased the [Ca2+]
i
by 7.2%. Cells in a Ca2+-free buffer increased [Ca2+]
i
after addition of 10 mm Ca2+, and this increase was abolished by nifedipine in both depolarizing and normal medium but not by ω-conotoxin. Single cell
experiments using video microscopy revealed that enterocytes remained polarized several hours after preparation and that the
Ca2+ entry and extrusion occurred at specific and different regions of the enterocyte outer membrane. Fluorescent staining of
L-type Ca2+ channels in the intestinal mucosa showed the most intense staining at the brushborder membrane.
These results demonstrate the presence of voltage gated L-type Ca2+ channels in enterocytes from the Atlantic cod. The channels are mainly located at the apical side of the cells, and there
is a polarized uptake of Ca2+ into the enterocytes. This suggests that the L-type Ca2+ channels are involved in the transcellular Ca2+ entry into the enterocytes.
Received: 21 August 1997/Revised: 15 April 1998 相似文献
9.
Gerrit Smit Diny M. J. Tubbing Jan W. Kijne Ben J. J. Lugtenberg 《Archives of microbiology》1991,155(3):278-283
The first step in attachment of Rhizobiaceae cells to plant root hair tips is mediated by a Ca2+-dependent, Ca2+-binding protein, rhicadhesin. The possible role of Ca2+ in synthesis, anchoring and activity of rhicadhesin was investigated. Growth of Rhizobium leguminosarum biovar viciae cells under Ca2+-limitation was found to result in loss of attachment ability. Under these conditions, rhicadhesin could not be usolated from the bacterial cell surface, but was found to be excreted in the growth medium. Divalent ions appeared to be essential for the ability of purified rhicadhesin to inhibit attachment of R. leguminosarum biovar viciae cells to pea root hair tips. Calcium ions were found not to be involved in binding of rhicadhesin to the plant surface, but appeared to be involved in anchoring of the adhesin to the bacterial cell surface. A model for the role of Ca2+ in activity of rhicadhesin is presented. 相似文献
10.
Regulation of critical cellular functions, including Ca2+-dependent gene expression, is determined by the temporal and spatial aspects of agonist-induced Ca2+ signals. Stimulation of cells with physiological concentrations of agonists trigger increases [Ca2+]i due to intracellular Ca2+ release and Ca2+ influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca2+]i increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca2+]i increases in human submandibular gland (HSG) cells. However, only Orai1-mediated Ca2+ entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca2+ store depletion in these cells, it is not clear how the cells decode individual Ca2+ signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca2+]i signals and activation of NFAT in single cells. We report that Orai1-mediated Ca2+ entry generates [Ca2+]i oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca2+ entry generates sustained [Ca2+]i elevation at high [CCh] and contributes to frequency of [Ca2+]i oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca2+ dependent gene expression pathways, consistent with the different patterns of [Ca2+]i signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display “all-or-none” activation that is exclusively driven by local [Ca2+]i generated by Orai1, independent of global [Ca2+]i changes or TRPC1-mediated Ca2+ entry. In contrast, Ca2+ entry via TRPC1 primarily regulates NFκB-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca2+ signals following agonist stimulation of cells, which determine the functional specificity of the channels in activating different Ca2+-dependent gene expression pathways. 相似文献
11.
The magnitude and spatial localization of Ca2+, K+ and H+ fluxes in growing and non-growing Limnobium stoloniferum root hairs was determined using non-invasive, ion-selective vibrating microelectrodes. Both the spatial pattern and magnitude of the ionic flux was dependent on the particular ion in question. Both H+ and Ca2+ influx was localized almost exclusively to the tips of growing root hairs, suggesting that these fluxes may be involved in directing growth. Influx of K+ showed no distinct localization and uptake appeared uniform along the length of the root hair. Competitive inhibition of Ca2+ influx using a range of Mg+ concentrations indicated that the magnitude of the Ca2+ flux entering the root hair tip did not determine growth rate; however, the presence of Ca2+ on the external face of the membrane was implicit for root hair integrity. Aluminum proved to be a potent inhibitor of root hair growth. At an exogenous Al concentration of 20 M a complete blockage of Ca2+ influx into root hair tips was observed, suggesting that Al blockage of Ca2+ influx could be involved in Al toxicity. However, at a lower Al concentration (2 M), Ca2+ fluxes were unaffected while inhibition of growth was still observed along with a distinct swelling of the root hair tip. The swelling at the root hair tips was identical in appearance to that seen in the presence of microtubule inhibitors, suggesting that Al could influence a number of different sites at the plasma-membrane surface and within the cell. The possible role(s) of Ca2+ and H+ fluxes in directing tip growth are discussed. 相似文献
12.
Maitotoxin (MTX), a potent polyether marine biotoxin, induces Ca2+ entry in different mammalian cells by activation of Ca2+ channels. The identity and modulation of the MTX-activated Ca2+ entry pathway is not known. In this work, we show, for the first time, that glucose and lactate can modulate the excitability of spermatogenic cell MTX-activated Ca2+ channels. Physiological and pharmacological evidences indicate that glucose and lactate differentially affect MTX-activated Ca2+ entry mainly through changes that these substrates induce on intracellular Ca2+ stores and the concentration of intracellular Ca2+ ([Ca2+]i) in spermatogenic cells. Our findings strongly suggest that MTX-activated Ca2+ channels in spermatogenic cells can be regulated by a Ca2+-CaM-dependent protein kinase. 相似文献
13.
14.
15.
《Channels (Austin, Tex.)》2013,7(1):62-75
Small conductance Ca2+-sensitive potassium (SK2) channels are voltage-independent, Ca2+-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca2+ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca2+ and Ca2+-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca2+ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses. 相似文献
16.
Jeong Hee Hong Qin Li Min Seuk Kim Dong Min Shin Stefan Feske Lutz Birnbaumer Kwong Tai Cheng Indu S. Ambudkar Shmuel Muallem 《Traffic (Copenhagen, Denmark)》2011,12(2):232-245
Polarized Ca2+ signals in secretory epithelial cells are determined by compartmentalized localization of Ca2+ signaling proteins at the apical pole. Recently the ER Ca2+ sensor STIM1 (stromal interaction molecule 1) and the Orai channels were shown to play a critical role in store‐dependent Ca2+ influx. STIM1 also gates the transient receptor potential‐canonical (TRPC) channels. Here, we asked how cell stimulation affects the localization, recruitment and function of the native proteins in polarized cells. Inhibition of Orai1, STIM1, or deletion of TRPC1 reduces Ca2+ influx and frequency of Ca2+ oscillations. Orai1 localization is restricted to the apical pole of the lateral membrane. Surprisingly, cell stimulation does not lead to robust clustering of native Orai1, as is observed with expressed Orai1. Unexpectedly, cell stimulation causes polarized recruitment of native STIM1 to both the apical and lateral regions, thus to regions with and without Orai1. Accordingly, STIM1 and Orai1 show only 40% colocalization. Consequently, STIM1 shows higher colocalization with the basolateral membrane marker E‐cadherin than does Orai1, while Orai1 showed higher colocalization with the tight junction protein ZO1. TRPC1 is expressed in both apical and basolateral regions of the plasma membrane. Co‐IP of STIM1/Orai1/IP3 receptors (IP3Rs)/TRPCs is enhanced by cell stimulation and disrupted by 2‐aminoethoxydiphenyl borate (2APB). The polarized localization and recruitment of these proteins results in preferred Ca2+ entry that is initiated at the apical pole. These findings reveal that in addition to Orai1, STIM1 likely regulates other Ca2+ permeable channels, such as the TRPCs. Both channels contribute to the frequency of [Ca2+] oscillations and thus impact critical cellular functions. 相似文献
17.
During vertebrate locomotion, spinal neurons act as oscillators when initiated by glutamate release from descending systems. Activation of NMDA receptors initiates Ca2+-mediated intrinsic membrane potential oscillations in central pattern generator (CPG) neurons. NMDA receptor-dependent intrinsic oscillations require Ca2+-dependent K+ (KCa2) channels for burst termination. However, the location of Ca2+ entry mediating KCa2 channel activation, and type of Ca2+ channel – which includes NMDA receptors and voltage-gated Ca2+ channels (VGCCs) – remains elusive. NMDA receptor-dependent Ca2+ entry necessitates presynaptic release of glutamate, implying a location at active synapses within dendrites, whereas VGCC-dependent Ca2+ entry is not similarly constrained. Where Ca2+ enters relative to KCa2 channels is crucial to information processing of synaptic inputs necessary to coordinate locomotion. We demonstrate that Ca2+ permeating NMDA receptors is the dominant source of Ca2+ during NMDA-dependent oscillations in lamprey spinal neurons. This Ca2+ entry is synaptically located, NMDA receptor-dependent, and sufficient to activate KCa2 channels at excitatory interneuron synapses onto other CPG neurons. Selective blockade of VGCCs reduces whole-cell Ca2+ entry but leaves membrane potential and Ca2+ oscillations unaffected. Furthermore, repetitive oscillations are prevented by fast, but not slow, Ca2+ chelation. Taken together, these results demonstrate that KCa2 channels are closely located to NMDA receptor-dependent Ca2+ entry. The close spatial relationship between NMDA receptors and KCa2 channels provides an intrinsic mechanism whereby synaptic excitation both excites and subsequently inhibits ventral horn neurons of the spinal motor system. This places the components necessary for oscillation generation, and hence locomotion, at glutamatergic synapses. 相似文献
18.
《Cell calcium》2020
Neuroendocrine adrenal chromaffin cells release neurohormones catecholamines in response to Ca2+ entry via voltage-gated Ca2+ channels (VGCCs). Adrenal chromaffin cells also express non-voltage-gated channels, which may conduct Ca2+ at negative membrane potentials, whose role in regulation of exocytosis is poorly understood. We explored how modulation of Ca2+ influx at negative membrane potentials affects basal cytosolic Ca2+ concentration ([Ca2+]i) and exocytosis in metabolically intact voltage-clamped bovine adrenal chromaffin cells. We found that in these cells, Ca2+ entry at negative membrane potentials is balanced by Ca2+ extrusion by the Na+/Ca2+ exchanger and that this balance can be altered by membrane hyperpolarization or stimulation with an inflammatory hormone bradykinin. Membrane hyperpolarization or application of bradykinin augmented Ca2+-carrying current at negative membrane potentials, elevated basal [Ca2+]i, and facilitated synchronous exocytosis evoked by the small amounts of Ca2+ injected into the cell via VGCCs (up to 20 pC). Exocytotic responses evoked by the injections of the larger amounts of Ca2+ via VGCCs (> 20 pC) were suppressed by preceding hyperpolarization. In the absence of Ca2+ entry via VGCCs and Ca2+ extrusion via the Na+/Ca2+ exchanger, membrane hyperpolarization induced a significant elevation in [Ca2+]i and asynchronous exocytosis. Our results indicate that physiological interferences, such as membrane hyperpolarization and/or activation of non-voltage-gated Ca2+ channels, modulate basal [Ca2+]i and, consequently, segregation of exocytotic vesicles and their readiness to be released spontaneously and in response to Ca2+ entry via VGCCs. These mechanisms may play role in homeostatic plasticity of neuronal and endocrine cells. 相似文献
19.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2019,1866(7):1037-1045
The intracellular calcium signaling processes are tightly regulated to ensure the generation of calcium signals with the specific spatiotemporal characteristics required for regulating various cell functions. Compartmentalization of the molecular components involved in the generation of these signals at discrete intracellular sites ensures the signaling specificity and transduction fidelity of the signal for regulating downstream effector processes. Store-operated calcium entry (SOCE) is ubiquitously present in cells and is critical for essential cell functions in a variety of tissues. SOCE is mediated via plasma membrane Ca2+ channels that are activated when luminal [Ca2+] of the endoplasmic reticulum ([Ca2+]ER) is decreased. The ER-resident stromal interaction molecules, STIM1 and STIM2, respond to decreases in [Ca2+]ER by undergoing conformational changes that cause them to aggregate at the cell periphery in ER-plasma membrane (ER-PM) junctions. At these sites, STIM proteins recruit Orai1 channels and trigger their activation. Importantly, the two STIM proteins concertedly modulate Orai1 function as well as the sensitivity of SOCE to ER-Ca2+ store depletion. Another family of plasma membrane Ca2+ channels, known as the Transient Receptor Potential Canonical (TRPC) channels (TRPC1-7) also contribute to sustained [Ca2+]i elevation. Although Ca2+ signals generated by these channels overlap with those of Orai1, they regulate distinct functions in the cells. Importantly, STIM1 is also required for plasma membrane localization and activation of some TRPCs. In this review, we will discuss various molecular components and factors that govern the activation, regulation and modulation of the Ca2+ signal generated by Ca2+ entry pathways in response to depletion of ER-Ca2+ stores. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. 相似文献