scholarly journals Functional Differences in Ionic Regulation between Alternatively Spliced Isoforms of the Na+-Ca2+ Exchanger from Drosophila melanogaster

1998 ◽  
Vol 111 (5) ◽  
pp. 691-702 ◽  
Author(s):  
Alexander Omelchenko ◽  
Christopher Dyck ◽  
Mark Hnatowich ◽  
John Buchko ◽  
Debora A. Nicoll ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Alternative Splicing ◽  
Xenopus Laevis Oocytes ◽  
Patch Clamp Technique ◽  
Inactive State ◽  
Functional Differences ◽  
Dependent Inactivation ◽  
Current Voltage ◽  
Alternatively Spliced ◽  
Regulatory Properties

Ion transport and regulation were studied in two, alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster. These exchangers, designated CALX1.1 and CALX1.2, differ by five amino acids in a region where alternative splicing also occurs in the mammalian Na+-Ca2+ exchanger, NCX1. The CALX isoforms were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the giant, excised patch clamp technique. Outward Na+-Ca2+ exchange currents, where pipette Ca2+o exchanges for bath Na+i, were examined in all cases. Although the isoforms exhibited similar transport properties with respect to their Na+i affinities and current–voltage relationships, significant differences were observed in their Na+i- and Ca2+i-dependent regulatory properties. Both isoforms underwent Na+i-dependent inactivation, apparent as a time-dependent decrease in outward exchange current upon Na+i application. We observed a two- to threefold difference in recovery rates from this inactive state and the extent of Na+i-dependent inactivation was approximately twofold greater for CALX1.2 as compared with CALX1.1. Both isoforms showed regulation of Na+-Ca2+ exchange activity by Ca2+i, but their responses to regulatory Ca2+i differed markedly. For both isoforms, the application of cytoplasmic Ca2+i led to a decrease in outward exchange currents. This negative regulation by Ca2+i is unique to Na+-Ca2+ exchangers from Drosophila, and contrasts to the positive regulation produced by cytoplasmic Ca2+ for all other characterized Na+-Ca2+ exchangers. For CALX1.1, Ca2+i inhibited peak and steady state currents almost equally, with the extent of inhibition being ≈80%. In comparison, the effects of regulatory Ca2+i occurred with much higher affinity for CALX1.2, but the extent of these effects was greatly reduced (≈20–40% inhibition). For both exchangers, the effects of regulatory Ca2+i occurred by a direct mechanism and indirectly through effects on Na+i-induced inactivation. Our results show that regulatory Ca2+i decreases Na+i-induced inactivation of CALX1.2, whereas it stabilizes the Na+i-induced inactive state of CALX1.1. These effects of Ca2+i produce striking differences in regulation between CALX isoforms. Our findings indicate that alternative splicing may play a significant role in tailoring the regulatory profile of CALX isoforms and, possibly, other Na+-Ca2+ exchange proteins.

scholarly journals Ionic Regulatory Properties of Brain and Kidney Splice Variants of the Ncx1 Na+–Ca2+ Exchanger

1999 ◽  
Vol 114 (5) ◽  
pp. 701-711 ◽  
Author(s):  
Chris Dyck ◽  
Alexander Omelchenko ◽  
Chadwick L. Elias ◽  
Beate D. Quednau ◽  
Kenneth D. Philipson ◽  
...  
Keyword(s):  
Splice Variants ◽  
Xenopus Laevis Oocytes ◽  
Low Concentrations ◽  
Dependent Inactivation ◽  
Current Voltage ◽  
Inward Currents ◽  
Alternatively Spliced ◽  
Experimental Protocols ◽  
Regulatory Properties ◽  
Insight Into

Ion transport and regulation of Na+–Ca2+ exchange were examined for two alternatively spliced isoforms of the canine cardiac Na+–Ca2+ exchanger, NCX1.1, to assess the role(s) of the mutually exclusive A and B exons. The exchangers examined, NCX1.3 and NCX1.4, are commonly referred to as the kidney and brain splice variants and differ only in the expression of the BD or AD exons, respectively. Outward Na+–Ca2+ exchange activity was assessed in giant, excised membrane patches from Xenopus laevis oocytes expressing the cloned exchangers, and the characteristics of Na+i- (i.e., I1) and Ca2+i- (i.e., I2) dependent regulation of exchange currents were examined using a variety of experimental protocols. No remarkable differences were observed in the current–voltage relationships of NCX1.3 and NCX1.4, whereas these isoforms differed appreciably in terms of their I1 and I2 regulatory properties. Sodium-dependent inactivation of NCX1.3 was considerably more pronounced than that of NCX1.4 and resulted in nearly complete inhibition of steady state currents. This novel feature could be abolished by proteolysis with α-chymotrypsin. It appears that expression of the B exon in NCX1.3 imparts a substantially more stable I1 inactive state of the exchanger than does the A exon of NCX1.4. With respect to I2 regulation, significant differences were also found between NCX1.3 and NCX1.4. While both exchangers were stimulated by low concentrations of regulatory Ca2+i, NCX1.3 showed a prominent decrease at higher concentrations (>1 μM). This does not appear to be due solely to competition between Ca2+i and Na+i at the transport site, as the Ca2+i affinities of inward currents were nearly identical between the two exchangers. Furthermore, regulatory Ca2+i had only modest effects on Na+i-dependent inactivation of NCX1.3, whereas I1 inactivation of NCX1.4 could be completely eliminated by Ca2+i. Our results establish an important role for the mutually exclusive A and B exons of NCX1 in modulating the characteristics of ionic regulation and provide insight into how alternative splicing tailors the regulatory properties of Na+–Ca2+ exchange to fulfill tissue-specific requirements of Ca2+ homeostasis.

scholarly journals Trans-acting Factors Required for Inclusion of Regulated Exons in the Ultrabithorax mRNAs of Drosophila melanogaster

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1517-1529 ◽  
Author(s):  
James M Burnette ◽  
Allyson R Hatton ◽  
A Javier Lopez
Keyword(s):  
Drosophila Melanogaster ◽  
Alternative Splicing ◽  
P Element ◽  
Splicing Factors ◽  
Loss Of Function ◽  
Large Collection ◽  
Splicing Pattern ◽  
Alternatively Spliced ◽  
Hypomorphic Alleles

Abstract Alternatively spliced Ultrabithorax mRNAs differ by the presence of internal exons mI and mII. Two approaches were used to identify trans-acting factors required for inclusion of these cassette exons. First, mutations in a set of genes implicated in the control of other alternative splicing decisions were tested for dominant effects on the Ubx alternative splicing pattern. To identify additional genes involved in regulation of Ubx splicing, a large collection of deficiencies was tested first for dominant enhancement of the haploinsufficient Ubx haltere phenotype and second for effects on the splicing pattern. Inclusion of the cassette exons in Ubx mRNAs was reduced strongly in heterozygotes for hypomorphic alleles of hrp48, which encodes a member of the hnRNP A/B family and is implicated in control of P-element splicing. Significant reductions of mI and mII inclusion were also observed in heterozygotes for loss-of-function alleles of virilizer, fl(2)d, and crooked neck. The products of virilizer and fl(2)d are also required for Sxl autoregulation at the level of splicing; crooked neck encodes a protein with structural similarities to yeast-splicing factors Prp39p and Prp42p. Deletion of at least five other loci caused significant reductions in the inclusion of mI and/or mII. Possible roles of identified factors are discussed in the context of the resplicing strategy for generation of alternative Ubx mRNAs.

Alternatively spliced isoform of apical Na+-K+-Cl− cotransporter gene encodes a furosemide-sensitive Na+-Cl−cotransporter

2001 ◽  
Vol 280 (4) ◽  
pp. F574-F582 ◽  
Author(s):  
Consuelo Plata ◽  
Patricia Meade ◽  
Amy Hall ◽  
Rick C. Welch ◽  
Norma Vázquez ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Alternative Splicing ◽  
Immunohistochemical Analysis ◽  
Xenopus Laevis Oocytes ◽  
Thick Ascending Limb ◽  
Terminal Domain ◽  
Medullary Thick Ascending Limb ◽  
Slc12a1 Gene ◽  
Alternatively Spliced ◽  
Oocyte Cytoplasm

In the absence of vasopressin, medullary thick ascending limb cells express a K+-independent, furosemide-sensitive Na+-Cl− cotransporter that is inhibited by hypertonicity. The murine renal specific Na+-K+-2 Cl− cotransporter gene ( SLC12A1) gives rise to six alternatively spliced isoforms. Three feature a long COOH-terminal domain that encodes the butmetanide-sensitive Na+-K+-2 Cl−cotransporter (BSC1–9/NKCC2), and three with a short COOH-terminal domain, known as mBSC1-A4, B4, or F4 (19). Here we have determined the functional characteristics of mBSC1-A4, as expressed in Xenopus laevis oocytes. When incubated at normal oocyte osmolarity (∼200 mosmol/kgH2O), mBSC1–4-injected oocytes do not express significant Na+ uptake over H2O-injected controls, and immunohistochemical analysis shows that the majority of mBSC1–4 protein is in the oocyte cytoplasm and not at the plasma membrane. In contrast, when mBSC1–4 oocytes are exposed to hypotonicity (∼100 mosmol/kgH2O), a significant increase in Na+uptake but not in 86Rb+ uptake is observed. The increased Na+ uptake is Cl− dependent, furosemide sensitive, and cAMP sensitive but K+independent. Sodium uptake increases with decreasing osmolarity between 120 and 70 mosmol/kgH2O ( r = 0.95, P < 0.01). Immunohistochemical analysis shows that in hypotonic conditions mBSC1-A4 protein is expressed in the plasma membrane. These studies indicate that the mBSC1-A4 isoform of the SLC12A1 gene encodes a hypotonically activated, cAMP- and furosemide-sensitive Na+-Cl− cotransporter. Thus it is possible that alternative splicing of the BSC1 gene could provide the molecular mechanism enabling the Na+-Cl−-to-Na+-K+-2Cl−switching in thick ascending limb cells.

scholarly journals The Drosophila melanogaster tropomyosin II gene produces multiple proteins by use of alternative tissue-specific promoters and alternative splicing.

1988 ◽  
Vol 8 (9) ◽  
pp. 3591-3602 ◽  
Author(s):  
P D Hanke ◽  
R V Storti
Keyword(s):  
Drosophila Melanogaster ◽  
Alternative Splicing ◽  
Gene Evolution ◽  
Cdna Clones ◽  
Developmentally Regulated ◽  
Dna Coding ◽  
Tropomyosin Isoforms ◽  
Alternatively Spliced ◽  
Transcriptional Units ◽  
Regulated Promoters

The structure of the Drosophila melanogaster tropomyosin II (TmII) gene has been determined by DNA sequencing of cDNA clones and the genomic DNA coding for the gene. Two overlapping transcriptional units produce at least four different tropomyosin isoforms. A combination of developmentally regulated promoters and alternative splicing produces both muscle and cytoskeletal tropomyosin isoforms. One promoter is a muscle-specific promoter and produces three different tropomyosin isoforms by alternative splicing of the last three 3' exons. The second promoter has the characteristics of a housekeeping promoter and produces a cytoskeletal tropomyosin isoform. Several internal exons along with a final 3' exon are alternatively spliced in the cytoskeletal transcript. The intron-exon boundaries of the TmII gene are identical to the intron-exon boundaries of all vertebrate tropomyosin genes reported, but are very different from the intron-exon boundaries of the D. melanogaster tropomyosin I gene. The TmII gene is the only reported tropomyosin gene that has two promoters and a quadruple alternative splice choice for the final exon. Models for the mechanism of D. melanogaster tropomyosin gene evolution are discussed.

scholarly journals Developmental and cell-specific expression ofCacna1dsplice variants

2019 ◽  
Author(s):  
LaCarubba Brianna ◽  
Bunda Alexandra ◽  
Savage Kitty ◽  
Sargent Hannah ◽  
Akiki Marie ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Brain Development ◽  
Splice Variants ◽  
Cell Types ◽  
Adult Brain ◽  
Specific Expression ◽  
C Terminus ◽  
Dependent Inactivation ◽  
Alternatively Spliced ◽  
Domain I

ABSTRACTCaV1.3 is an L-type voltage-gated calcium channel implicated in several functions including gene expression, pacemaking activity, and neurotransmitter release. The gene that encodes the CaVα1-pore forming subunit of CaV1.3 (Cacna1d) is a multi-exon gene that undergoes extensive alternative splicing, which provides functional versatility to this gene across tissues and cell-types. The function and expression of severalCacna1dsplice variants within the C-terminus have been previously characterized. These splice variants differ in their voltage-dependence of activation, Ca2+-dependent inactivation, and their sensitivity to dihydropyridines. However, less is known about alternatively spliced exons inCacna1dlocated downstream of domain I and upstream of the C-terminus (e11, e22a/e22, e31a/e31b/e32). Here, we performed a systematic study to determine the developmental and cell-specific expression of severalCacna1dsplice variants. We found that the cassette e11 is upregulated during brain development, and in adult cortical tissue is more abundant in excitatory neurons relative to inhibitory interneurons. This exon is also upregulated upon nerve growth factor (NGF) induced differentiation of pheochromocytoma cells, PC12. At the functional level, the splice variants resulting from e11 alternative splicing (+e11-Cacna1dand Δe11-Cacna1d) form functional CaV1.3 channels with similar biophysical properties in expression mammalian systems. Of the pair of mutually exclusive exons, e22a and e22, the later dominates at all stages. However, we observed a slight upregulation of e22 from embryonic to adult human brain. A second pair of mutually exclusive exons, e31a and e31b, was also studied. We found that e31a increases during brain development. Finally, the cassette exon 32 is repressed in adult brain tissue.

scholarly journals Differential Modulation of SERCA2 Isoforms by Calreticulin

1998 ◽  
Vol 142 (4) ◽  
pp. 963-973 ◽  
Author(s):  
Linu M. John ◽  
James D. Lechleiter ◽  
Patricia Camacho
Keyword(s):  
Endoplasmic Reticulum ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Xenopus Laevis Oocytes ◽  
Differential Modulation ◽  
Functional Differences ◽  
Selective Targeting ◽  
Inositol 1,4,5 Trisphosphate ◽  
Alternatively Spliced ◽  
Wave Properties

In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca2+ oscillations in a manner consistent with inhibition of Ca2+ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA)2 gene display differential Ca2+ wave properties and sensitivity to modulation by calreticulin. We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences. Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding. In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.

scholarly journals Functional differences between flounder and rat thiazide-sensitive Na-Cl cotransporter

2002 ◽  
Vol 282 (4) ◽  
pp. F599-F607 ◽  
Author(s):  
Norma Vázquez ◽  
Adriana Monroy ◽  
Elisa Dorantes ◽  
Rosario A. Muñoz-Clares ◽  
Gerardo Gamba
Keyword(s):  
Phorbol Esters ◽  
Xenopus Laevis Oocytes ◽  
Kinase C ◽  
Functional Differences ◽  
Low Concentrations ◽  
Biphasic Effect ◽  
Uptake Medium ◽  
Regulatory Properties ◽  
Menten Constant

The purpose of the present study was to determine the major functional, pharmacological, and regulatory properties of the flounder thiazide-sensitive Na-Cl cotransporter (flTSC) to make a direct comparison with our recent characterization of the rat TSC (rTSC; Monroy A, Plata C, Hebert SC, and Gamba G. Am J Physiol Renal Physiol 279: F161–F169, 2000). When expressed in Xenopus laevis oocytes, flTSC exhibits lower affinity for Na+ than for Cl−, with apparent Michaelis-Menten constant ( K m) values of 58.2 ± 7.1 and 22.1 ± 4.2 mM, respectively. These K m values are significantly higher than those observed in rTSC. The Na+ and Cl− affinities decreased when the concentration of the counterion was lowered, suggesting that the binding of one ion increases the affinity of the transporter for the other. The effect of several thiazides on flTSC function was biphasic. Low concentrations of thiazides (10−9 to 10−7 M) resulted in activation of the cotransporter, whereas higher concentrations (10−6 to 10−4 M) were inhibitory. In rTSC, this biphasic effect was observed only with chlorthalidone. The affinity for thiazides in flTSC was lower than in rTSC, but the affinity in flTSC was not affected by the Na+ or the Cl− concentration in the uptake medium. In addition to thiazides, flTSC and rTSC were inhibited by Hg2+, with an apparent higher affinity for rTSC. Finally, flTSC function was decreased by activation of protein kinase C with phorbol esters and by hypertonicity. In summary, we have found significant regulatory, kinetic, and pharmacological differences between flTSC and rTSC orthologues.

The Drosophila melanogaster tropomyosin II gene produces multiple proteins by use of alternative tissue-specific promoters and alternative splicing

1988 ◽  
Vol 8 (9) ◽  
pp. 3591-3602
Author(s):  
P D Hanke ◽  
R V Storti
Keyword(s):  
Drosophila Melanogaster ◽  
Alternative Splicing ◽  
Gene Evolution ◽  
Cdna Clones ◽  
Developmentally Regulated ◽  
Dna Coding ◽  
Tropomyosin Isoforms ◽  
Alternatively Spliced ◽  
Transcriptional Units ◽  
Regulated Promoters

The structure of the Drosophila melanogaster tropomyosin II (TmII) gene has been determined by DNA sequencing of cDNA clones and the genomic DNA coding for the gene. Two overlapping transcriptional units produce at least four different tropomyosin isoforms. A combination of developmentally regulated promoters and alternative splicing produces both muscle and cytoskeletal tropomyosin isoforms. One promoter is a muscle-specific promoter and produces three different tropomyosin isoforms by alternative splicing of the last three 3' exons. The second promoter has the characteristics of a housekeeping promoter and produces a cytoskeletal tropomyosin isoform. Several internal exons along with a final 3' exon are alternatively spliced in the cytoskeletal transcript. The intron-exon boundaries of the TmII gene are identical to the intron-exon boundaries of all vertebrate tropomyosin genes reported, but are very different from the intron-exon boundaries of the D. melanogaster tropomyosin I gene. The TmII gene is the only reported tropomyosin gene that has two promoters and a quadruple alternative splice choice for the final exon. Models for the mechanism of D. melanogaster tropomyosin gene evolution are discussed.

scholarly journals Splicing-associated chromatin signatures: a combinatorial and position-dependent role for histone marks in splicing definition

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
E. Agirre ◽  
A. J. Oldfield ◽  
N. Bellora ◽  
A. Segelle ◽  
R. F. Luco
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Embryonic Stem ◽  
Chromatin Modifications ◽  
Histone Marks ◽  
Splicing Regulators ◽  
Machine Learning Approach ◽  
Alternatively Spliced ◽  
Rna Motif ◽  
Regulation Changes

AbstractAlternative splicing relies on the combinatorial recruitment of splicing regulators to specific RNA binding sites. Chromatin has been shown to impact this recruitment. However, a limited number of histone marks have been studied at a global level. In this work, a machine learning approach, applied to extensive epigenomics datasets in human H1 embryonic stem cells and IMR90 foetal fibroblasts, has identified eleven chromatin modifications that differentially mark alternatively spliced exons depending on the level of exon inclusion. These marks act in a combinatorial and position-dependent way, creating characteristic splicing-associated chromatin signatures (SACS). In support of a functional role for SACS in coordinating splicing regulation, changes in the alternative splicing of SACS-marked exons between ten different cell lines correlate with changes in SACS enrichment levels and recruitment of the splicing regulators predicted by RNA motif search analysis. We propose the dynamic nature of chromatin modifications as a mechanism to rapidly fine-tune alternative splicing when necessary.

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