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.

Plasma-membrane calcium pumps and hereditary deafness

2007 ◽  
Vol 35 (5) ◽  
pp. 913-918 ◽  
Author(s):  
M. Brini ◽  
F. Di Leva ◽  
T. Domi ◽  
L. Fedrizzi ◽  
D. Lim ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Plasma Membrane ◽  
Alternative Splicing ◽  
Membrane Domains ◽  
Hereditary Deafness ◽  
Calcium Pumps ◽  
Specific Targeting ◽  
Alternatively Spliced ◽  
The Central Nervous System

In mammals, four different genes encode four PMCA (plasma-membrane Ca2+-ATPase) isoforms. PMCA1 and 4 are expressed ubiquitously, and PMCA2 and 3 are expressed predominantly in the central nervous system. More than 30 variants are generated by mechanisms of alternative splicing. The physiological meaning of the existence of so many isoforms is not clear, but evidently it must be related to the cell-specific demands of Ca2+ homoeostasis. Recent studies suggest that the alternatively spliced regions in PMCA are responsible for specific targeting to plasma membrane domains, and proteins that bind specifically to the pumps could contribute to further regulation of Ca2+ control. In addition, the combination of proteins obtained by alternative splicing occurring at two different sites could be responsible for different functional characteristics of the pumps.

Functional comparison of renal Na-K-Cl cotransporters between distant species

2003 ◽  
Vol 284 (2) ◽  
pp. C365-C370 ◽  
Author(s):  
Édith Gagnon ◽  
Biff Forbush ◽  
Luc Caron ◽  
Paul Isenring
Keyword(s):  
Alternative Splicing ◽  
Transmembrane Domain ◽  
Splice Variants ◽  
Xenopus Laevis Oocytes ◽  
Thick Ascending Limb ◽  
Kinetic Behavior ◽  
Transport Activity ◽  
Conserved Residues ◽  
Spatially Distributed ◽  
The Relationship

In the shark (sa), two variants of the renal Na-K-Cl cotransporter (saNKCC2A and saNKCC2F) are produced by alternative splicing of the second transmembrane domain (tm2). In mammals, these splice variants, as well as a third variant (NKCC2B), are spatially distributed along the thick ascending limb of Henle and exhibit divergent kinetic behaviors. To test whether different tm2 in saNKCC2 are also associated with different kinetic phenotypes, we examined the ion dependence of86Rb influx for shark and rabbit splice variants expressed in Xenopus laevis oocytes. We found that, in both species, A forms have higher cation affinities than F forms. In regard to Cl affinity, however, the A-F difference was more pronounced in rabbit, and the relationship between transport activity and Cl concentration was not always sigmoidal. These results show that the tm2of saNKCC2 is, as in rabbit, important for Cl transport, and they suggest that the ability of the distal NKCC2-expressing segment to extract Cl from the luminal fluid differs among species. We have also found that the renal NKCC2 of distant vertebrates share similar affinities for cations. This finding points to the existence of highly conserved residues that mediate the kinetic behavior of the NKCC2 splice variants.

Control of H(+)-HCO3- plasma membrane transporters by urea hyperosmolality in rat medullary thick ascending limb

1994 ◽  
Vol 266 (5) ◽  
pp. C1157-C1164 ◽  
Author(s):  
F. Leviel ◽  
M. Froissart ◽  
H. Soualmia ◽  
J. Poggioli ◽  
M. Paillard ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Calcium Concentration ◽  
Inhibitory Concentration ◽  
Initial Rate ◽  
Maximum Velocity ◽  
Membrane Transporters ◽  
Thick Ascending Limb ◽  
Michaelis Constant ◽  
Medullary Thick Ascending Limb ◽  
Experimental Protocols

Hyperosmolality inhibits bicarbonate absorption by the rat medullary thick ascending limb (MTAL) by unknown mechanisms. Intracellular pH (pHi) was monitored with use of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in rat MTAL tubule suspensions to specify the H(+)-HCO3- membrane transporters affected by hyperosmolality. Measurements were made after > or = 15-min incubation of the cells in media rendered hypertonic by urea to avoid any change in cell volume. Na(+)-H+ antiport activity, estimated from the Na(+)-induced initial rate of pHi recovery of Na(+)-depleted acidified cells in the presence of 0.1 mM furosemide to inhibit Na(+)-K(+)-2Cl- cotransport, was inhibited by 300 mM urea and 10(-8) M arginine vasopressin (AVP) in an additive manner. Na(+)-H+ antiport inhibition by urea hyperosmolality was maximal at 300 mM urea with a half-maximal inhibitory concentration of 75 mM and was due to a 28% decrease in maximum velocity (Vmax) with no effect on the Michaelis constant for sodium. Urea hyperosmolality (300 mM) did not affect steady-state intracellular calcium concentration ([Ca2+]i), assessed with use of fura 2 fluorescence, and still inhibited Na(+)-H+ antiport in MTAL cells loaded with 1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid to minimize any transient change in [Ca2+]i during the preincubation in urea medium. Furthermore, 300 mM urea did not stimulate basal or AVP-induced adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Plasma membrane H(+)-adenosinetriphosphatase (ATPase) activity and HCO3- transport, assessed by appropriate experimental protocols, were unaltered by 300 mM urea.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Study of calmodulin binding to the alternatively spliced C-terminal domain of the plasma membrane calcium pump

Biochemistry ◽  
1992 ◽  
Vol 31 (47) ◽  
pp. 11785-11792 ◽  
Author(s):  
Felix Kessler ◽  
Rocco Falchetto ◽  
Roger Heim ◽  
Ruedi Meili ◽  
Thomas Vorherr ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Calcium Pump ◽  
Calmodulin Binding ◽  
Terminal Domain ◽  
Plasma Membrane Calcium Pump ◽  
Alternatively Spliced

scholarly journals Alternative splicing converts STIM2 from an activator to an inhibitor of store-operated calcium channels

2015 ◽  
Vol 209 (5) ◽  
pp. 653-670 ◽  
Author(s):  
Anshul Rana ◽  
Michelle Yen ◽  
Amir Masoud Sadaghiani ◽  
Seth Malmersjö ◽  
Chan Young Park ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Alternative Splicing ◽  
Calcium Channels ◽  
Potent Inhibitor ◽  
Cellular Functions ◽  
Functional Flexibility ◽  
Allosteric Interaction ◽  
Store Operated Calcium Entry ◽  
Positive Regulators ◽  
Alternatively Spliced

Store-operated calcium entry (SOCE) regulates a wide variety of essential cellular functions. SOCE is mediated by STIM1 and STIM2, which sense depletion of ER Ca2+ stores and activate Orai channels in the plasma membrane. Although the amplitude and dynamics of SOCE are considered important determinants of Ca2+-dependent responses, the underlying modulatory mechanisms are unclear. In this paper, we identify STIM2β, a highly conserved alternatively spliced isoform of STIM2, which, in contrast to all known STIM isoforms, is a potent inhibitor of SOCE. Although STIM2β does not by itself strongly bind Orai1, it is recruited to Orai1 channels by forming heterodimers with other STIM isoforms. Analysis of STIM2β mutants and Orai1-STIM2β chimeras suggested that it actively inhibits SOCE through a sequence-specific allosteric interaction with Orai1. Our results reveal a previously unrecognized functional flexibility in the STIM protein family by which alternative splicing creates negative and positive regulators of SOCE to shape the amplitude and dynamics of Ca2+ signals.

scholarly journals RNF141 interacts with KRAS to promote colorectal cancer progression

Oncogene ◽  
2021 ◽  
Author(s):  
Jiuna Zhang ◽  
Xiaoyu Jiang ◽  
Jie Yin ◽  
Shiying Dou ◽  
Xiaoli Xie ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Plasma Membrane ◽  
Tumor Growth ◽  
Cancer Progression ◽  
Critical Role ◽  
Ring Finger ◽  
Immunohistochemical Analysis ◽  
Bimolecular Fluorescence Complementation

AbstractRING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC.

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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