Role of RYR3 splice variants in calcium signaling in mouse nonpregnant and pregnant myometrium

Alternative splicing of ryanodine receptor subtype 3 (RYR3) may generate a short isoform (RYR3S) without channel function and a functional full-length isoform (RYR3L). The RYR3S isoform has been shown to negatively regulate the native RYR2 subtype in smooth muscle cells as well as the RYR3L isoform when both isoforms were coexpressed in HEK-293 cells. Mouse myometrium expresses only the RYR3 subtype, but the role of RYR3 isoforms obtained by alternative splicing and their activation by cADP-ribose during pregnancy have never been investigated. Here, we show that both RYR3S and RYR3L isoforms are differentially expressed in nonpregnant and pregnant mouse myometrium. The use of antisense oligonucleotides directed against each isoform indicated that only RYR3L was activated by caffeine and cADP-ribose in nonpregnant myometrium. These RYR3L-mediated Ca2+ releases were negatively regulated by RYR3S expression. At the end of pregnancy, the relative expression of RYR3L versus RYR3S and its ability to respond to cADP-ribose were increased. Therefore, our results suggest that physiological regulation of RYR3 alternative splicing may play an essential role at the end of pregnancy.

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Influence of intracellular Ca2+ and alternative splicing on the pharmacological profile of ANO1 channels

AJP Cell Physiology ◽

10.1152/ajpcell.00070.2016 ◽

2016 ◽

Vol 311 (3) ◽

pp. C437-C451 ◽

Cited By ~ 22

Author(s):

Tae Sik Sung ◽

Kate O'Driscoll ◽

Haifeng Zheng ◽

Nicholas J. Yapp ◽

Normand Leblanc ◽

...

Keyword(s):

Splice Variants ◽

Channel Blockers ◽

Anoctamin 1 ◽

Patch Clamp Technique ◽

Hek 293 ◽

Naturally Occurring ◽

293 Cells ◽

Intracellular Ca ◽

Inside Out

Anoctamin-1 (ANO1) is a Ca2+-activated Cl− channel expressed in many types of cells. Splice variants of ANO1 have been shown to influence the biophysical properties of conductance. It has been suggested that several new antagonists of ANO1 with relatively high affinity and selectivity might be useful for experimental and, potentially, therapeutic purposes. We investigated the effects of intracellular Ca2+ concentration ([Ca2+]i) at 100-1,000 nM, a concentration range that might be achieved in cells during physiological activation of ANO1 channels, on blockade of ANO1 channels expressed in HEK-293 cells. Whole cell and excised patch configurations of the patch-clamp technique were used to perform tests on a variety of naturally occurring splice variants of ANO1. Blockade of ANO1 currents with aminophenylthiazole (T16Ainh-A01) was highly dependent on [Ca2+]i. Increasing [Ca2+]i reduced the potency of this blocker. Similar Ca2+-dependent effects were also observed with benzbromarone. Experiments on excised, inside-out patches showed that the diminished potency of the blockers caused by intracellular Ca2+ might involve a competitive interaction for a common binding site or repulsion of the blocking drugs by electrostatic forces at the cytoplasmic surface of the channels. The degree of interaction between the channel blockers and [Ca2+]i depends on the splice variant expressed. These experiments demonstrate that the efficacy of ANO1 antagonists depends on [Ca2+]i, suggesting a need for caution when ANO1 blockers are used to determine the role of ANO1 in physiological functions and in their use as therapeutic agents.

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Assessment for the Role of Serotonin Receptor Subtype 3 for the Analgesic Action of Morphine at the Spinal Level

The Korean Journal of Pain ◽

10.3344/kjp.2005.18.2.113 ◽

2005 ◽

Vol 18 (2) ◽

pp. 113

Author(s):

Myung Ha Yoon ◽

Hong Buem Bae ◽

Jeong Il Choi ◽

Seok Jae Kim ◽

Chang Mo Kim ◽

...

Keyword(s):

Serotonin Receptor ◽

Receptor Subtype ◽

Analgesic Action ◽

Spinal Level ◽

Subtype 3

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Species-Specific Regulation of TRPM2 by PI(4,5)P2 via the Membrane Interfacial Cavity

International Journal of Molecular Sciences ◽

10.3390/ijms22094637 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4637

Author(s):

Daniel Barth ◽

Andreas Lückhoff ◽

Frank J. P. Kühn

Keyword(s):

Amino Acid Residues ◽

Hek 293 ◽

Complete Inactivation ◽

293 Cells ◽

Activation Mechanisms ◽

Specific Regulation ◽

Species Specific ◽

Inside Out ◽

Positively Charged

The human apoptosis channel TRPM2 is stimulated by intracellular ADR-ribose and calcium. Recent studies show pronounced species-specific activation mechanisms. Our aim was to analyse the functional effect of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), commonly referred to as PIP2, on different TRPM2 orthologues. Moreover, we wished to identify the interaction site between TRPM2 and PIP2. We demonstrate a crucial role of PIP2, in the activation of TRPM2 orthologues of man, zebrafish, and sea anemone. Utilizing inside-out patch clamp recordings of HEK-293 cells transfected with TRPM2, differential effects of PIP2 that were dependent on the species variant became apparent. While depletion of PIP2 via polylysine uniformly caused complete inactivation of TRPM2, restoration of channel activity by artificial PIP2 differed widely. Human TRPM2 was the least sensitive species variant, making it the most susceptible one for regulation by changes in intramembranous PIP2 content. Furthermore, mutations of highly conserved positively charged amino acid residues in the membrane interfacial cavity reduced the PIP2 sensitivity in all three TRPM2 orthologues to varying degrees. We conclude that the membrane interfacial cavity acts as a uniform PIP2 binding site of TRPM2, facilitating channel activation in the presence of ADPR and Ca2+ in a species-specific manner.

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Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

Anesthesiology ◽

10.1097/00000542-200512000-00009 ◽

2005 ◽

Vol 103 (6) ◽

pp. 1156-1166 ◽

Cited By ~ 7

Author(s):

Kevin J. Gingrich ◽

Son Tran ◽

Igor M. Nikonorov ◽

Thomas J. Blanck

Keyword(s):

Charge Transfer ◽

Calcium Channels ◽

Dependent Manner ◽

Current Time ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Dependent Inactivation ◽

Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

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Phosphoregulation of the intracellular termini of K+-Cl− cotransporter 2 (KCC2) enables flexible control of its activity

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.004349 ◽

2018 ◽

Vol 293 (44) ◽

pp. 16984-16993 ◽

Cited By ~ 6

Author(s):

Antje Cordshagen ◽

Wiebke Busch ◽

Michael Winklhofer ◽

Hans Gerd Nothwang ◽

Anna-Maria Hartmann

Keyword(s):

Intrinsic Activity ◽

Hek 293 ◽

Flexible Control ◽

Mediated Effects ◽

293 Cells ◽

Bona Fide ◽

Graded Activity ◽

First Time ◽

Increased Activity

The pivotal role of K+-Cl− cotransporter 2 (KCC2) in inhibitory neurotransmission and severe human diseases fosters interest in understanding posttranslational regulatory mechanisms such as (de)phosphorylation. Here, the regulatory role of the five bona fide phosphosites Ser31, Thr34, Ser932, Thr999, and Thr1008 was investigated by the use of alanine and aspartate mutants. Tl+-based flux analyses in HEK-293 cells demonstrated increased transport activity for S932D (mimicking phosphorylation) and T1008A (mimicking dephosphorylation), albeit to a different extent. Increased activity was due to changes in intrinsic activity, as it was not caused by increased cell-surface abundance. Substitutions of Ser31, Thr34, or Thr999 had no effect. Additionally, we show that the indirect actions of the known KCC2 activators staurosporine and N-ethylmaleimide (NEM) involved multiple phosphosites. S31D, T34A, S932A/D, T999A, or T1008A/D abrogated staurosporine mediated stimulation, and S31A, T34D, or S932D abolished NEM-mediated stimulation. This demonstrates for the first time differential effects of staurosporine and NEM on KCC2. In addition, the staurosporine-mediated effects involved both KCC2 phosphorylation and dephosphorylation with Ser932 and Thr1008 being bona fide target sites. In summary, our data reveal a complex phosphoregulation of KCC2 that provides the transporter with a toolbox for graded activity and integration of different signaling pathways.

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Anoctamin 5/TMEM16E facilitates muscle precursor cell fusion

The Journal of General Physiology ◽

10.1085/jgp.201812097 ◽

2018 ◽

Vol 150 (11) ◽

pp. 1498-1509 ◽

Cited By ~ 14

Author(s):

Jarred M. Whitlock ◽

Kuai Yu ◽

Yuan Yuan Cui ◽

H. Criss Hartzell

Keyword(s):

Cell Fusion ◽

Ionic Current ◽

Ionic Currents ◽

Muscle Physiology ◽

Muscle Repair ◽

Hek 293 ◽

293 Cells ◽

Phospholipid Scrambling ◽

Muscle Precursor Cell

Limb-girdle muscular dystrophy type 2L (LGMD2L) is a myopathy arising from mutations in ANO5; however, information about the contribution of ANO5 to muscle physiology is lacking. To explain the role of ANO5 in LGMD2L, we previously hypothesized that ANO5-mediated phospholipid scrambling facilitates cell–cell fusion of mononucleated muscle progenitor cells (MPCs), which is required for muscle repair. Here, we show that heterologous overexpression of ANO5 confers Ca2+-dependent phospholipid scrambling to HEK-293 cells and that scrambling is associated with the simultaneous development of a nonselective ionic current. MPCs isolated from adult Ano5−/− mice exhibit defective cell fusion in culture and produce muscle fibers with significantly fewer nuclei compared with controls. This defective fusion is associated with a decrease of Ca2+-dependent phosphatidylserine exposure on the surface of Ano5−/− MPCs and a decrease in the amplitude of Ca2+-dependent outwardly rectifying ionic currents. Viral introduction of ANO5 in Ano5−/− MPCs restores MPC fusion competence, ANO5-dependent phospholipid scrambling, and Ca2+-dependent outwardly rectifying ionic currents. ANO5-rescued MPCs produce myotubes having numbers of nuclei similar to wild-type controls. These data suggest that ANO5-mediated phospholipid scrambling or ionic currents play an important role in muscle repair.

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Extracellular signal-regulated kinase activation by parathyroid hormone in distal tubule cells

AJP Renal Physiology ◽

10.1152/ajprenal.00288.2006 ◽

2007 ◽

Vol 292 (3) ◽

pp. F1028-F1034 ◽

Cited By ~ 26

Author(s):

W. Bruce Sneddon ◽

Yanmei Yang ◽

Jianming Ba ◽

Lisa M. Harinstein ◽

Peter A. Friedman

Keyword(s):

Conditioned Media ◽

Kidney Cells ◽

Wild Type ◽

Egfr Transactivation ◽

Hek 293 ◽

Erk Activation ◽

Hek 293 Cells ◽

293 Cells ◽

Extracellular Signal

The PTH receptor (PTH1R) activates multiple signaling pathways, including extracellular signal-regulated kinases 1 and 2 (ERK1/2). The role of epidermal growth factor receptor (EGFR) transactivation in ERK1/2 activation by PTH in distal kidney cells, a primary site of PTH action, was characterized. ERK1/2 phosphorylation was stimulated by PTH and blocked by the EGFR inhibitor, AG1478. Upon PTH stimulation, metalloprotease cleavage of membrane-bound heparin-binding fragment (HB-EGF) induced EGFR transactivation of ERK. Conditioned media from PTH-treated distal kidney cells activated ERK in HEK-293 cells. AG1478 added to HEK-293 cells ablated transactivation by conditioned media. HB-EGF directly activated ERK1/2 in HEK-293 cells. Pretreatment of distal kidney cells with the metalloprotease inhibitor GM-6001 abolished transactivation of ERK1/2 by PTH. The role of the PTH1R COOH terminus in PTX-sensitive ERK1/2 activation was characterized in HEK-293 cells transfected with wild-type PTH1R, with a PTH1R mutated at its COOH terminus, or with PTH1R truncated at position 480. PTH stimulated ERK by wild-type, mutated and truncated PTH1Rs 21-, 27- and 57-fold, respectively. Thus, the PTH1R COOH terminus exerts an inhibitory effect on ERK activation. EBP50, a scaffolding protein that binds to the PDZ recognition domain of the PTH1R, impaired PTH but not isoproterenol or calcitonin-induced ERK activation. Pertussis toxin inhibited PTH-stimulated ERK1/2 by mutated and truncated PTH1Rs and abolished ERK1/2 activation by wild-type PTH1R. We conclude that ERK phosphorylation in distal kidney cells by PTH requires PTH1R activation of Gi, which leads to stimulation of metalloprotease-mediated cleavage of HB-EGF and transactivation of the EGFR and is regulated by EBP50.

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Splice variants of a ClC-2 chloride channel with differing functional characteristics

AJP Cell Physiology ◽

10.1152/ajpcell.2000.279.4.c1198 ◽

2000 ◽

Vol 279 (4) ◽

pp. C1198-C1210 ◽

Cited By ~ 47

Author(s):

L. Pablo Cid ◽

María-Isabel Niemeyer ◽

Alfredo Ramírez ◽

Francisco V. Sepúlveda

Keyword(s):

Splice Variants ◽

Donor Site ◽

Proximal Colon ◽

Site Directed Mutagenesis ◽

Intestinal Epithelial ◽

Chloride Currents ◽

Hek 293 ◽

Internal Donor ◽

293 Cells ◽

Functional Consequences

We identified two ClC-2 clones in a guinea pig intestinal epithelial cDNA library, one of which carries a 30-bp deletion in the NH2 terminus. PCR using primers encompassing the deletion gave two products that furthermore were amplified with specific primers confirming their authenticity. The corresponding genomic DNA sequence gave a structure of three exons and two introns. An internal donor site occurring within one of the exons accounts for the deletion, consistent with alternative splicing. Expression of the variants gpClC-2 and gpClC-2Δ77–86 in HEK-293 cells generated inwardly rectifying chloride currents with similar activation characteristics. Deactivation, however, occurred with faster kinetics in gpClC-2Δ77–86. Site-directed mutagenesis suggests that a protein kinase C-mediated phosphorylation consensus site lost in gpClC-2Δ77–86 is not responsible for the observed change. The deletion-carrying variant is found in most tissues examined, and it appears more abundant in proximal colon, kidney, and testis. The presence of a splice variant of ClC-2 modified in its NH2-terminal domain could have functional consequences in tissues where their relative expression levels are different.

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