scholarly journals Two PKC consensus sites on human acid-sensing ion channel 1b differentially regulate its function

2009 ◽  
Vol 296 (2) ◽  
pp. C372-C384 ◽  
Author(s):  
Edlira Bashari ◽  
Yawar J. Qadri ◽  
Zhen-Hong Zhou ◽  
Niren Kapoor ◽  
Susan J. Anderson ◽  
...  
Keyword(s):  
Ion Channel ◽  
Lipid Bilayers ◽  
Glioma Cells ◽  
Surface Expression ◽  
Wild Type ◽  
Open Probability ◽  
Cation Conductance ◽  
Human Acid ◽  
Electrode Voltage ◽  
Pkc Activation

Human acid-sensing ion channel 1b (hASIC1b) is a H+-gated amiloride-sensitive cation channel. We have previously shown that glioma cells exhibit an amiloride-sensitive cation conductance. Amiloride and the ASIC1 blocker psalmotoxin-1 decrease the migration and proliferation of glioma cells. PKC also abolishes the amiloride-sensitive conductance of glioma cells and inhibits hASIC1b open probability in planar lipid bilayers. In addition, hASIC1b's COOH terminus has been shown to interact with protein interacting with C kinase (PICK)1, which targets PKC to the plasma membrane. Therefore, we tested the hypothesis that PKC regulation of hASIC1b at specific PKC consensus sites inhibits hASIC1b function. We mutated three consensus PKC phosphorylation sites (T26, S40, and S499) in hASIC1b to alanine, to prevent phosphorylation, and to glutamic acid or aspartic acid, to mimic phosphorylation. Our data suggest that S40 and S499 are critical sites mediating the modulation of hASIC1b by PKC. We expressed mutant hASIC1b constructs in Xenopus oocytes and measured acid-activated currents by two-electrode voltage clamp. T26A and T26E did not exhibit acid-activated currents. S40A was indistinguishable from wild type (WT), whereas S40E, S499A, and S499D currents were decreased. The PKC activators PMA and phorbol 12,13-dibutyrate inhibited WT hASIC1b and S499A, and PMA had no effect on S40A or on WT hASIC1b in oocytes pretreated with the PKC inhibitor chelerythrine. Chelerythrine inhibited WT hASIC1b and S40A but had no effect on S499A or S40A/S499A. PKC activators or the inhibitor did not affect the surface expression of WT hASIC1b. These data show that the two PKC consensus sites S40 and S499 differentially regulate hASIC1b and mediate the effects of PKC activation or PKC inhibition on hASIC1b. This will result in a deeper understanding of PKC regulation of this channel in glioma cells, information that may help in designing potentially beneficial therapies in their treatment.

scholarly journals Acute Downregulation of ENaC by EGF Involves the PY Motif and Putative ERK Phosphorylation Site

2007 ◽  
Vol 130 (3) ◽  
pp. 313-328 ◽  
Author(s):  
Rebecca A. Falin ◽  
Calvin U. Cotton
Keyword(s):  
Kinase Inhibitor ◽  
Collecting Duct ◽  
Phosphorylation Site ◽  
Surface Expression ◽  
Wild Type ◽  
Open Probability ◽  
Erk Phosphorylation ◽  
C Terminus ◽  
Py Motif ◽  
Erk Kinase

The epithelial sodium channel (ENaC) is expressed in a variety of tissues, including the renal collecting duct, where it constitutes the rate-limiting step for sodium reabsorption. Liddle's syndrome is caused by gain-of-function mutations in the β and γ subunits of ENaC, resulting in enhanced Na reabsorption and hypertension. Epidermal growth factor (EGF) causes acute inhibition of Na absorption in collecting duct principal cells via an extracellular signal–regulated kinase (ERK)–dependent mechanism. In experiments with primary cultures of collecting duct cells derived from a mouse model of Liddle's disease (β-ENaC truncation), it was found that EGF inhibited short-circuit current (Isc) by 24 ± 5% in wild-type cells but only by 6 ± 3% in homozygous mutant cells. In order to elucidate the role of specific regions of the β-ENaC C terminus, Madin-Darby canine kidney (MDCK) cell lines that express β-ENaC with mutation of the PY motif (P616L), the ERK phosphorylation site (T613A), and C terminus truncation (R564stop) were created using the Phoenix retroviral system. All three mutants exhibited significant attenuation of the EGF-induced inhibition of sodium current. In MDCK cells with wild-type β-ENaC, EGF-induced inhibition of Isc (<30 min) was fully reversed by exposure to an ERK kinase inhibitor and occurred with no change in ENaC surface expression, indicative of an effect on channel open probability (Po). At later times (>30 min), EGF-induced inhibition of Isc was not reversed by an ERK kinase inhibitor and was accompanied by a decrease in ENaC surface expression. Our results are consistent with an ERK-mediated decrease in ENaC open probability and enhanced retrieval of sodium channels from the apical membrane.

Amiloride-sensitive Na+ channels contribute to regulatory volume increases in human glioma cells

2007 ◽  
Vol 293 (3) ◽  
pp. C1181-C1185 ◽  
Author(s):  
Sandra B. Ross ◽  
Catherine M. Fuller ◽  
James K. Bubien ◽  
Dale J. Benos
Keyword(s):  
Ion Channel ◽  
Potential Role ◽  
Glioma Cells ◽  
Brain Parenchyma ◽  
Volume Increase ◽  
Cation Conductance ◽  
Normal Human ◽  
Hyperosmolar Solutions ◽  
The Brain

Despite intensive research, brain tumors remain among the most difficult type of malignancies to treat, due largely to their diffusely invasive nature and the associated difficulty of adequate surgical resection. To migrate through the brain parenchyma and to proliferate, glioma cells must be capable of significant changes in shape and volume. We have previously reported that glioma cells express an amiloride- and psalmotoxin-sensitive cation conductance that is not found in normal human astrocytes. In the present study, we investigated the potential role of this ion channel to mediate regulatory volume increase in glioma cells. We found that the ability of the cells to volume regulate subsequent to cell shrinkage by hyperosmolar solutions was abolished by both amiloride and psalmotoxin 1. This toxin is thought to be a specific peptide inhibitor of acid-sensing ion channel (ASIC1), a member of the Deg/ENaC superfamily of cation channels. We have previously shown this toxin to be an effective blocker of the glioma cation conductance. Our data suggest that one potential role for this conductance may be to restore cell volume during the cell's progression thorough the cell cycle and while the tumor cell migrates within the interstices of the brain.

scholarly journals Gain-of-function variant of the human epithelial sodium channel

2013 ◽  
Vol 304 (2) ◽  
pp. F207-F213 ◽  
Author(s):  
Jingxin Chen ◽  
Thomas R. Kleyman ◽  
Shaohu Sheng
Keyword(s):  
Single Channel ◽  
Expression System ◽  
Surface Expression ◽  
Wild Type ◽  
Open Probability ◽  
Gain Of Function ◽  
Human Disorders ◽  
Human Genome Sequencing ◽  
Xenopus Oocyte Expression ◽  
Salt Sensitive Hypertension

Epithelial Na+ channel (ENaC) mutations are associated with several human disorders, underscoring the importance of these channels in human health. Recent human genome sequencing projects have revealed a large number of ENaC gene variations, several of which have been found in individuals with salt-sensitive hypertension, cystic fibrosis, and other disorders. However, the functional consequences of most variants are unknown. In this study, we used the Xenopus oocyte expression system to examine the functional properties of a human ENaC variant. Oocytes expressing αβγL511Q human ENaCs showed 4.6-fold greater amiloride-sensitive currents than cells expressing wild-type channels. The γL511Q variant did not significantly alter channel surface expression. Single channel recordings revealed that the variant had fourfold higher open probability than wild type. In addition, γL511Q largely eliminated the Na+ self-inhibition response, which reflects a downregulation of ENaC open probability by extracellular Na+. Moreover, γL511Q diminished chymotrypsin-induced activation of the mutant channel. We conclude that γL511Q is a gain-of-function human ENaC variant. Our results suggest that γL511Q enhances ENaC activity by increasing channel open probability and dampens channel regulation by extracellular Na+ and proteases.

scholarly journals The Amino-Terminal Region of Vpr from Human Immunodeficiency Virus Type 1 Forms Ion Channels and Kills Neurons

1999 ◽  
Vol 73 (5) ◽  
pp. 4230-4238 ◽  
Author(s):  
S. C. Piller ◽  
G. D. Ewart ◽  
D. A. Jans ◽  
P. W. Gage ◽  
G. B. Cox
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Cell Death ◽  
Ion Channels ◽  
Ion Channel ◽  
Lipid Bilayers ◽  
Planar Lipid Bilayers ◽  
Wild Type ◽  
Immunodeficiency Virus

ABSTRACT We have previously reported that the accessory protein Vpr from human immunodeficiency virus type 1 forms cation-selective ion channels in planar lipid bilayers and is able to depolarize intact cultured neurons by causing an inward sodium current, resulting in cell death. In this study, we used site-directed mutagenesis and synthetic peptides to identify the structural regions responsible for the above functions. Mutations in the N-terminal region of Vpr were found to affect channel activity, whereas this activity was not affected by mutations in the hydrophobic region of Vpr (amino acids 53 to 71). Analysis of mutants containing changes in the basic C terminus confirmed previous results that this region, although not necessary for ion channel function, was responsible for the observed rectification of wild-type Vpr currents. A peptide comprising the first 40 N-terminal amino acids of Vpr (N40) was found to be sufficient to form ion channels similar to those caused by wild-type Vpr in planar lipid bilayers. Furthermore, N40 was able to cause depolarization of the plasmalemma and cell death in cultured hippocampal neurons with a time course similar to that seen with wild-type Vpr, supporting the idea that this region is responsible for Vpr ion channel function and cytotoxic effects. Since Vpr is found in the serum and cerebrospinal fluids of AIDS patients, these results may have significance for AIDS pathology.

scholarly journals Deletion of α-subunit exon 11 of the epithelial Na+ channel reveals a regulatory module

2014 ◽  
Vol 306 (5) ◽  
pp. F561-F567 ◽  
Author(s):  
Jingxin Chen ◽  
Thomas R. Kleyman ◽  
Shaohu Sheng
Keyword(s):  
Surface Expression ◽  
Significant Loss ◽  
Na Channel ◽  
Channel Activity ◽  
Wild Type ◽  
Open Probability ◽  
Α Subunit ◽  
Regulatory Module ◽  
Channel Expression ◽  
Exon 11

Epithelial Na+ channel (ENaC) subunits (α, β, and γ) found in functional complexes are translated from mature mRNAs that are similarly processed by the inclusion of 13 canonical exons. We examined whether individual exons 3–12, encoding the large extracellular domain, are required for functional channel expression. Human ENaCs with an in-frame deletion of a single α-subunit exon were expressed in Xenopus oocytes, and their functional properties were examined by two-electrode voltage clamp. With the exception of exon 11, deletion of an individual exon eliminated channel activity. Channels lacking α-subunit exon 11 were hyperactive. Oocytes expressing this mutant exhibited fourfold greater amiloride-sensitive whole cell currents than cells expressing wild-type channels. A parallel fivefold increase in channel open probability was observed with channels lacking α-subunit exon 11. These mutant channels also exhibited a lost of Na+ self-inhibition, whereas we found similar levels of surface expression of mutant and wild-type channels. In contrast, in-frame deletions of exon 11 from either the β- or γ-subunit led to a significant loss of channel activity, in association with a marked decrease in surface expression. Our results suggest that exon 11 within the three human ENaC genes encodes structurally homologous yet functionally diverse domains and that exon 11 in the α-subunit encodes a module that regulates channel gating.

scholarly journals Human TRPA1 is an inherently mechanosensitive bilayer-gated ion channel

2020 ◽  
Author(s):  
Lavanya Moparthi ◽  
Peter M. Zygmunt
Keyword(s):  
Ion Channel ◽  
Lipid Bilayers ◽  
Transient Receptor Potential ◽  
Single Channel ◽  
Applied Pressure ◽  
Mechanical Stimuli ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Pressure Interval

AbstractThe Transient Receptor Potential Ankyrin 1 (TRPA1) channel is an intrinsic chemo- and thermo-sensitive ion channel with distinct sensory signaling properties. Although a role of TRPA1 in mammalian mechanosensory transduction in vivo seems likely, it remains to be shown that TRPA1 has the inherent capability to respond to mechanical stimuli. Here we have used the patch-clamp technique to study the response of human purified TRPA1 (hTRPA1), reconstituted into artificial lipid bilayers, to changes in bilayer pressure. We report that hTRPA1 responded with increased single-channel open probability (Po) within the applied pressure interval of 7.5 to 60 mmHg with a half maximum Po (P50) value of 38.0 ± 2.3 mmHg. The Po value reached a maximum close to 1 (0.87 ± 0.02) at 60 mmHg. Within the same pressure interval, hTRPA1 without its N-terminal ankyrin repeat domain (Δ1-688 hTRPA1) responded fully opened (0.99 ± 0.01) at 60 mmHg and with a P50 value of 39.0 ± 1.1 mmHg. The pressure-evoked responses of hTRPA1 and Δ1-688 hTRPA1 at 45 mmHg were inhibited by the TRPA1 antagonist HC030031, and the activity of purified hTRPA1 at 45 mmHg was abolished by the thiol reducing agent tris(2-carboxyethyl)phosphine (TCEP). In conclusion, hTRPA1 is an inherent mechanosensitive ion channel gated by force-from-lipids. The hTRPA1 mechanosensitivity is dependent on the redox environment, and it is suggested that oxidative stress shifts hTRPA1 into a protein conformation sensitive to mechanical stimuli.

scholarly journals Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle

2014 ◽  
Vol 306 (5) ◽  
pp. C460-C470 ◽  
Author(s):  
Kiril L. Hristov ◽  
Amy C. Smith ◽  
Shankar P. Parajuli ◽  
John Malysz ◽  
Georgi V. Petkov
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Guinea Pig ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Channel Regulation ◽  
Pkc Activation

Large-conductance voltage- and Ca2+-activated K+ (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca2+ imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca2+ sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca2+ levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca2+-dependent mechanism, thus increasing DSM contractility.

scholarly journals Enhanced Long-Term Stability for Single Ion Channel Recordings Using Suspended Poly(lipid) Bilayers

2009 ◽  
Vol 131 (19) ◽  
pp. 6662-6663 ◽  
Author(s):  
Benjamin A. Heitz ◽  
Juhua Xu ◽  
Henry K. Hall ◽  
Craig A. Aspinwall ◽  
S. Scott Saavedra
Keyword(s):  
Ion Channel ◽  
Lipid Bilayers ◽  
Term Stability ◽  
Long Term Stability

Cell cycle arrest and astrocytic differentiation resulting from PTEN expression in glioma cells

1999 ◽  
Vol 91 (5) ◽  
pp. 822-830 ◽  
Author(s):  
Jun-ichi Adachi ◽  
Katsumi Ohbayashi ◽  
Tomonari Suzuki ◽  
Tomio Sasaki
Keyword(s):  
Expression System ◽  
Biological Significance ◽  
Glioma Cells ◽  
Genetic Alterations ◽  
Pten Expression ◽  
Human Glioma ◽  
Wild Type ◽  
Content Type ◽  
Astrocytic Differentiation ◽  
Fine Print

Object. Genetic alterations of the PTEN gene (also known as MMAC1 or TEP1) have frequently been identified in high-grade gliomas, indicating that inactivation of PTEN plays a crucial role in human glioma progression. The aim of this study was to assess the biological significance of PTEN inactivation in the development of glioma.Methods. The authors introduced wild-type PTEN complementary DNA into four human glioma cell lines (T98G, U-251MG, U-87MG, and A172) containing endogenous aberrant PTEN alleles. The number of colonies transfected with the wild-type PTEN was reduced to 15 to 32% of those found after transfection of a control vector, suggesting growth suppression by the exogenous PTEN. To analyze phenotypic alterations produced by PTEN expression, T98G-derived clones with inducible PTEN expression were further established using a tetracycline-regulated inducible gene expression system. Induction of PTEN expression suppressed the in vitro growth of T98G cells with accumulation of G1 phase cells. Furthermore, when cells were cultured in the presence of the extracellular matrix (ECM), PTEN expression caused distinct morphological changes, with multiple and elongated cytoplasmic processes similar to those of normal astrocytes. The level of glial fibrillary acidic protein, an intermediate protein specifically expressed in differentiated astrocytes, was upregulated concomitantly.Conclusions. These findings strongly indicate that exogenous PTEN expression inhibits the proliferation of glioma cells by inducing G1 arrest and elicits astrocytic differentiation in the presence of the ECM. Inactivation of PTEN would play an important role in the enhancement of unregulated growth of undifferentiated glioma cells.

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