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1.
Skeletal muscle cells express different isoforms of the calcium channel α2/δ subunit 总被引:1,自引:0,他引:1
We examined developmental changes in calcium channel alpha2/delta subunit mRNA in skeletal muscle and their possible influence on L-type calcium currents (ICa-L). Several isoforms of alpha2/delta-1 mRNA were found in myotubes and muscle fibers, and their relative levels changed with time in culture or age of the animal. Levels of alpha2/delta-1a were largest in older myotubes and was the only alpha2/delta-1 isoform present in adult muscle. Both myotubes and muscle fibers also expressed low levels of alpha2/delta-2 and alpha2/delta-3 mRNA at all ages. alpha2/delta-4 mRNA could not be detected in either myotubes or muscle fibers. Changes in amplitude and voltage-dependent inactivation of the ICa-L concurred with the shift in alpha2/delta-1 isoform message, suggesting that alternative splicing of this subunit might be important for modulation of ICa-L. 相似文献
2.
García J 《American journal of physiology. Cell physiology》2011,301(1):C44-C52
The α2/δ1 and α(1)1.1 subunits are present at a 1:1 ratio in the dihydropyridine receptor (DHPR) from adult skeletal muscle. In contrast, during early myotube development α2/δ1 is present at higher levels than α(1)1.1 and localizes at the ends of the cells, suggesting that α2/δ1 may have a role independent from DHPRs. We sought to identify binding partners of α2/δ1 at a period when levels of α(1)1.1 are low. Analysis of protein complexes in their native configuration established that α2/δ1 may be associating with ATP5b, a subunit of a mitochondrial ATP synthase complex. This interaction was confirmed with fluorescence resonance energy transfer and coimmunoprecipitation. The association of α2/δ1 and ATP5b occurs in intracellular membranes and at the plasma membrane, where they form a functional signaling complex capable of accelerating the rate of decline of calcium transients. The acceleration of decay was more evident when myotubes were stimulated with a train of pulses. Our data indicate that the α2/δ1 subunit is not only part of the DHPR but that it may interact with other cellular components in developing myotubes, such as the ATP5b in its atypical localization in the plasma membrane. 相似文献
3.
Alicia M Ebert Catherine A McAnelly Ashok Srinivasan Rachel Lockridge Mueller David B Garrity Deborah M Garrity 《BMC molecular biology》2008,9(1):38
Background
Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary β subunits to chaperone the pore-forming α subunit to the plasma membrane, and to modulate channel electrophysiology [1]. Several β subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse β2, but not in the other three β family members, are embryonic lethal at E10.5 due to defects in cardiac contractility [2]. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of β subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of β2 subunits in zebrafish and other teleosts. 相似文献4.
《Gene》1997,186(2):299-304
β4 and α6 integrin subunits dimerize to form an adhesion receptor that is necessary to nucleate hemidesmosomes and to anchor epithelial cells to their basal laminae. β4 is also expressed in Schwann cells (which do not contain hemidesmosomes) in peripheral nerve, where it may function in the formation or maintenance of myelin. The cDNA for β4 integrin has been cloned from epithelia-derived human and mouse tissues. We cloned cDNAs encoding β4 integrin from libraries derived from rat peripheral nerve, and determined the complete nucleotide sequence encoding the signal peptide and mature protein. Comparison of the deduced amino acid (aa) sequence revealed 95.1% and 87.5% identity with the mouse and human epithelia-derived sequences, respectively. The amino acid sequence of postulated signal transduction domains in β4 was 100% identical among rat, mouse, and human. Our cDNA clones included two of the four postulated alternatively spliced variants previously described in epithelial clones. Despite the potentially diverse functions of β4 integrin in Schwann cells and keratinocytes, the cDNAs for nerve-derived β4 integrin are highly similar to those cloned from epithelia. 相似文献
5.
Polyclonal antiserum raised against the native PG1 isoform of tomato fruit (Lycopersicon esculentum Mill.) polygalacturonase [poly(1,4--d-galacturonide) glycanohydrolase, EC 3.2.1.15] bound to each of the subunits of the protein and also to a range of other fruit proteins. Affinity purification was used to remove antibody molecules that bound to the native form of the PG2 isoform. The resulting serum bound to native PG1, denatured PG2 and -subunits of PG1 but not to native PG2 or other fruit proteins. This anti-PG1 serum was used to monitor the occurrence of the PG1 -subunit and PG2 in detergent extracts of tomato tissues. The -subunit polypeptide was detected in pericarp but not locule tissue of fruit, including fruit of the rin and nor mutants. It increased in amount in the pericarp tissues from an early stage to the mature green stage, clearly prior to any appreciable accumulation of the PG2 subunit. The -subunit polypeptide was not detected in stem or leaf tissues. A PG2-specific antiserum was used to study the interaction of PG2 with the isolated -subunit. The PG2 isoform was bound to the -subunit over a wide range of salt concentrations and pH; the interaction was independent of the presence of reducing agents. It is concluded that strong non-covalent forces are involved in the interaction. The results are consistent with a model in which the -subunit is positioned in the cell wall structure and provides a specific binding site for the active PG2 subunit when this is synthesised during ripening.Abbreviations B
breaker
- MG
mature green
- Mr
relative molecular mass
-
nor
non-ripening mutant
- PAGE
polyacrylamide gel electrophoresis
- PG
polygalacturonase
-
rin
ripening inhibitor mutant
- SDS
sodium dodecyl sulphate 相似文献
6.
Zhou TT Zhang ZW Liu J Zhang JP Jiao BH 《International journal of biological sciences》2012,8(5):630-639
Aberrant protein glycosylation plays major roles in neurodegenerative diseases, including Parkinson's disease (PD). Glycoproteomics showed that the glycosylation of sodium channel β4 was significantly increased in human brain tissue. β4-specific antibodies reacted in immunoblot assays with the 35- and 38-kDa bands from the membrane fractions isolated from neonatal PD transgenic mice but only with the 35-kDa band of the neonatal wild-type mice. The size of the 38-kDa immunoreactive protein is in close agreement with previously reported, suggesting heavy glycosylation of this protein in adult wild-type and neonatal PD transgenic brain tissues. However, the neonatal wild-type mice membrane fractions only contained the 35-kDa immunoreactive protein, and the additional 38-kDa band was not shown until postnatal day 7. Enzymatic deglycosylation of the membrane preparations only converted the 38-kDa band into a faster migrating protein, which was consistent with heavy glycosylation of this protein. The glycosylated state of β4 was developmentally regulated and was altered in disease state. Neurite outgrowth assay demonstrated that overexpression of deglycosylated mutant β4-MUT accelerated neurite extension and increased the number of filopodia-like protrusions, when compared with β4-WT and the vector. These results suggest that extensive glycosylation of β4 subunit play roles in morphological changes, and the altered glycosylation may be involved in the pathogenesis of PD. 相似文献
7.
Murakami M Aoyama M Suzuki T Sasano H Nakayama S Iijima T 《Molecular and cellular biochemistry》2003,254(1-2):217-225
This study reports a novel splice variant form of the voltage-dependent calcium channel 2 subunit (2g). This variant is composed of the conserved amino-terminal sequences of the 2a subunit, but lacks the -subunit interaction domain (BID), which is thought essential for interactions with the 1 subunit. Gene structure analysis revealed that this gene was composed of 13 translated exons spread over 107 kb of the genome. The gene structure of the 2 subunit was similar in exon-intron organization to the murine 3 and human 4 subunits. Electrophysiological evaluation revealed that 2a and 2g affected channel properties in different ways. The 2a subunit increased the peak amplitude, but failed to increase channel inactivation, while 2g had no significant effects on either the peak current amplitude or channel inactivation. Other subunits, such as 3 and 4, significantly increased the peak current and accelerated current inactivation. 相似文献
8.
9.
Integrin heterodimers play diverse and important roles in physiological and pathological processes, such as cell adhesion, migration, proliferation, differentiation, angiogenesis, and tumor progression, via the outside-in and/or inside-out signaling pathways. Aberrant functions of integrins have been implicated in the causation and intervention of multiple diseases. Integrin β(4), a laminin-5 (LN5) receptor, mainly locates in the adhesion structure of hemidesmosome (HD). Most of the previous researches concentrated on the role of integrin β(4) in cancer and cancer therapy, and a few focused on the physiological roles of normal mammalian cells. Recently, accumulating data reveal that integrin β(4) participates in cell death, macroautophagy (hereafter autophagy), senescence, and differentiation regulations in various cell types including human umbilical vein endothelial cells (HUVECs), mesenchymal stem cells, and mouse neural cells, implying the key roles of integrin β(4) in the physiological alteration of mammalian cells. Thus, the elucidation of integrin β(4)-mediated signaling may undoubtedly contribute to novel therapeutic strategies for various human diseases, such as vascular and neural disorders. We have reviewed the roles of integrin β(4) in neural cells. In the present review we will discuss the recent research progress in the inherent functions and pharmacological modulation of integrin β(4) in vascular endothelial cells. 相似文献
10.
11.
Dysfunction of airway smooth muscle (ASM) is an essential feature of airway remodeling in chronic asthma. However, the precise mechanisms of this pathological process have not been well studied. In previous study, we found that β1-integrin, which was dramatically upregulated in ASM cells in an asthmatic mouse model, was associated with the cell proliferation. In this study, we employed short hairpin RNA (shRNA) targeting β1-integrin to assess the effect of down-regulation of this receptor on the proliferative aspects and migratory properties of ASM cells in vitro. The cells were treated with shRNA expression vectors directed against β1-integrin, control vectors that included the blank control, empty vector without shRNA, and mismatched shRNA, respectively. The mRNA and protein expressions of β1-integrin were determined by real-time PCR and Western blotting. Cell proliferation was measured by BrdU ELISA and cell cycle by fluorescence-activated cell sorter. Cell apoptosis was detected by Annexin V-PE/7-AAD staining. Cell migration assays were evaluated by transwell assay and expression of IL-6 and IL-8 by ELISA. The results revealed that shRNA targeting β1-integrin significantly decreased the mRNA and protein expressions of β1-integrin, enhanced the proportion of cells in G0/G1 phase, decreased the proportion in S phase, promoted cell apoptosis, inhibited cell proliferation, migration, IL-6 and IL-8 secretion in vitro. In conclusion, the overexpression of β1-integrin in ASM cells is essential for airway dysfunction development because it promotes proliferative aspects and migratory properties of ASM cells. Importantly, shRNA targeting β1-integrin may provide a new approach to preventing airway remodeling in chronic asthma. 相似文献
12.
《Channels (Austin, Tex.)》2013,7(1):26-40
In skeletal muscle, L-type calcium channels (DHPRs), localized to plasma membrane sarcoplasmic reticulum junctions, are tightly packed into groups of four termed tetrads. Here, we have used bimolecular fluorescence complementation (BiFC) and targeted biotinylation to probe the structure and organization of β1a subunits associated with native CaV1.1 in DHPRs of myotubes. The construct YN-β1a-YC, in which the non-fluorescent fragments of YFP (“YN” corresponding to YFP residues 1–158, and “YC” corresponding to YFP residues 159–238) were fused, respectively, to the N- and C-termini of β1a, was fully functional and displayed yellow fluorescence within DHPR tetrads after expression in β1-knockout (β1KO) myotubes; this yellow fluorescence demonstrated the occurrence of BiFC of YN and YC on the β1a N- and C-termini. In these experiments, we avoided overexpression because control experiments in non-muscle cells indicated that this could result in non-specific BiFC. BiFC of YN-β1a-YC in DHPR tetrads appeared to be intramolecular between N- and C-termini of individual β1a subunits rather than between adjacent DHPRs because BiFC (1) was observed for YN-β1a-YC co-expressed with CaV1.2 (which does not form tetrads) and (2) was not observed after co-expression of YN-β1a-YN plus YC-β1a-YC in β1KO myotubes. Thus, β1a function is compatible with N- and C-termini being close enough together to allow BiFC. However, both termini appeared to have positional freedom and not to be closely opposed by other junctional proteins since both were accessible to gold-streptavidin conjugates. Based on these results, a model is proposed for the arrangement of β1a subunits in DHPR tetrads. 相似文献
13.
14.
Modulation of the Ca- and voltage-dependent K channel—KCa—by receptors coupled to the G proteins G
i
/G
o
and G
s
has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches somatostatin (somatotropin-releasing inhibitory factor; SRIF) caused concentration-dependent inhibition of the KCa channel, an effect that was prevented by pertussis toxin (PTX). In inside-out patches, exogenous subunits of either G
i
or G
o
-type G proteins also inhibited the KCa channel (IC50 5.9 and 5.7 pM, respectively). These data indicate that SRIF suppresses KCa channel activity via a membrane-delimited pathway that involves the subunits of PTX-sensitive G proteins G
i
and/or G
o
. In outside-out patches, activation of G
s
either by -agonists or with cholera toxin (CTX) increased KCa channel activity, consistent with a membrane-delimited stimulatory pathway linking the -adrenergic receptor to the KCa channel via G
s
. In outside-out patches, channel inhibition by SRIF suppressed the stimulatory effect of -agonists but not that of CTX, while in inside-out patches CTX reversed channel inhibition induced by exogenous
i
or
o
. Taken together these data suggest that KCa channel activity is enhanced by activation of G
s
and blocked by activated G
i
and/or G
o
. Further, KCa channel stimulation by activated G
s
may be direct, while inhibition by G
i
/G
o
may involve deactivation of G
s
. In inside-out patches KCa channel activity was reduced by an activator of protein kinase C (PKC) and enhanced by inhibitors of PKC, indicating that PKC also acts to inhibit the KCa channel via a membrane delimited pathway. In outside-out patches, chelerythrine, a membrane permeant inhibitor of PKC prevented the inhibitory effect of SRIF, and in inside-out patches PKC inhibitors prevented the inhibitory effect of exogenous
i
or
o
. These data indicate that PKC facilitates the inhibitory effect of the PTX-sensitive G proteins which are activated by coupling to SRIF receptors. To account for these results a mechanism is proposed whereby PKC may be involved in G
i
/G
o
-induced deactivation of G
s
.The authors would like to thank Dr. S. Ciani for many helpful discussions, Dr. A.E. Boyd III for supplying the HIT cells, Drs. J. Codina and L. Birnbaumer for supplying the alpha subunits of the G proteins G
i
and G
o
, and Mrs. Satoko Hagiwara for preparing and maintaining the cell cultures.This work was supported by grant DCB-8919368 from the National Science Foundation and a research grant (W-P 880513) from the American Diabetes Association to B.R., and by grant RO1-DK39652 from the National Institutes of Health to G.T.E. 相似文献
15.
Qin Miao Huang Yuji Masuda Takashi Takahashi 《Biochemical and biophysical research communications》2010,391(1):542-8416
Mammalian DNA polymerase δ (pol δ) is essential for DNA replication, though the functions of this smallest subunit of POLD4 have been elusive. We investigated pol δ activities in vitro and found that it was less active in the absence of POLD4, irrespective of the presence of the accessory protein PCNA. shRNA-mediated reduction of POLD4 resulted in a marked decrease in colony formation activity by Calu6, ACC-LC-319, and PC-10 cells. We also found that POLD4 reduction was associated with an increased population of karyomere-like cells, which may be an indication of DNA replication stress and/or DNA damage. The karyomere-like cells retained an ability to progress through the cell cycle, suggesting that POLD4 reduction induces modest genomic instability, while allowing cells to grow until DNA damage reaches an intolerant level. Our results indicate that POLD4 is required for the in vitro pol δ activity, and that it functions in cell proliferation and maintenance of genomic stability of human cells. 相似文献
16.
John A. Brown Gerald P. Jahreis Richard T. Swank 《Biochemical and biophysical research communications》1981,99(2):691-699
The presence of a precursor form of β-glucuronidase, with a subunit molecular weight of 75,000 was demonstrated in mouse kidney. This was later processed to the mature form, with subunit molecular weight of 71,500. Tissue fractionation revealed that the precursor was associated with the microsomes whereas the mature form was associated with the lysosomes. In mice lacking egasyn both forms of β-glucuronidase were present, but the rate of processing was elevated compared to normal. 相似文献
17.
Young S. Kim Ze Wang Robert M. Levin Samuel Chacko 《Molecular and cellular biochemistry》1994,131(2):115-124
The obstruction of the bladder outlet induces a marked increase in bladder mass, and this is accompanied by reduced contractility of bladder smooth muscle and alteration in the cellular architecture. In this study, we show that the composition of various isoforms of actin, a major component of the contractile apparatus and the cytoskeletal structure of smooth muscle, is altered in response to the obstruction-induced bladder hypertrophy. Northern blot analysis of the total RNA isolated from hypertrophied urinary bladder muscle, using a cDNA probe specific for smooth muscle -actin, shows over 200% increase in the -actin mRNA. However, the estimate of the amount of actin from the 2D gel reveals only a 16% increase in -actin, since the 2D gel electrophoresis does not distinguish -smooth muscle actin from -cytoplasmic actin. The bladder smooth muscle -actin and the smooth muscle -actin mRNA are not altered in response to the hypertrophy. The obstructed bladder also reveals a decrease in the -cytoplasmic actin (37%) and a concomitant diminution in the -cytoplasmic actin mRNA (29%). Hence, the composition of the actin isoforms in bladder smooth muscle is altered in response to the obstruction-induced hypertrophy. This alteration of the actin isoforms is observed at both the protein and mRNA levels. 相似文献
18.
19.
Juan Pablo Petiti Silvina Gutiérrez Jorge Humberto Mukdsi Ana Lucía De Paul Alicia Inés Torres 《Journal of molecular histology》2009,40(5-6):417-425
The variations of the intracellular localization of the individual protein kinase C (PKC) isoforms are related with their different biological functions. In this study, we have investigated the precise intracellular translocation of endogenous PKCα and PKCε in PMA-stimulated normal and tumoral lactotroph cells by using confocal and immunogold electron microscopy, which was correlated with the rate of cell proliferation of both pituitary cell phenotypes. The present results showed that the short phorbol ester incubation stimulated the proliferation of normal and tumoral lactotroph cells, as determined by the measurement of the BrdU-labelling index. The translocation of PKCα to plasma and nuclear membranes induced by PMA was more marked than that observed for PKCε in normal and tumoral lactotroph cells. Our results showed that PKCs translocation to the plasma and nuclear membranes varied from isozyme to isozyme emphasizing that PKCα could be related with the mitogenic stimulus exerted by phorbol ester. These data support the notion that specific PKC isozymes may exert spatially defined effects by virtue of their directed translocation to distinct intracellular sites. 相似文献
20.
Roland S. Wu Guoxia Liu Sergey I. Zakharov Neelesh Chudasama Howard Motoike Arthur Karlin Steven O. Marx 《The Journal of general physiology》2013,141(1):105-117
Large-conductance voltage- and Ca2+-gated K+ channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory β subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an ∼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in β2 and β3, as in β1 and β4, TM1 is closest to αS1 and αS2 and TM2 is closest to αS0. Mouse β3 (mβ3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of αS0–αS6. We identified by elimination mβ3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in β1. These are conserved in β2–β4, which have four additional Cys (eight in total), except that mβ3 has one fewer. In β1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In mβ3, the free Cys is at position 7; position 2 lacks a Cys present in all other β2–β4; and the disulfide pattern is 1–8, 3–4, and 5–6. Presumably, Cys 2 cross-links to Cys 7 in all other β2–β4. Cross-linking of mβ3 Cys152 to Cys substituted in the flanks of αS0–S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the αS6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of mβ3 perturbed. Although the mβ3 loop can move, its position with Cys152 near αK296, in which it blocks IbTX binding, is likely favored. 相似文献