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.

Increased focal Kv4.2 channel expression at the plasma membrane is the result of actin depolymerization

2004 ◽  
Vol 286 (2) ◽  
pp. H749-H759 ◽  
Author(s):  
Zhuren Wang ◽  
Jodene R. Eldstrom ◽  
Joshua Jantzi ◽  
Edwin D. Moore ◽  
David Fedida
Keyword(s):  
Actin Cytoskeleton ◽  
Single Channel ◽  
Expression System ◽  
Surface Expression ◽  
Cytochalasin D ◽  
Bright Spot ◽  
Open Probability ◽  
Hek Cells ◽  
Voltage Dependent ◽  
Channel Expression

Voltage-dependent potassium channel trafficking and localization are regulated by proteins of the cytoskeleton, but the mechanisms by which these occur are still unclear. Using human embryonic kidney (HEK) cells as a heterologous expression system, we tested the role of the actin cytoskeleton in modulating the function of Kv4.2 channels. Pretreatment (≥1 h) of HEK cells with 5 μM cytochalasin D to disrupt the actin microfilaments greatly augmented whole cell Kv4.2 currents at potentials positive to –20 mV. However, no changes in the voltage dependence of activation and inactivation of macroscopic currents were observed to account for this increase. Similarly, single channel recordings failed to reveal any significant changes in the single channel conductance, open probability, and kinetics. However, the mean patch current was increased from 0.9 ± 0.2 pA in control to 6.7 ± 3.0 pA in the presence of cytochalasin D. Imaging experiments revealed a clear increase in the surface expression of the channels and the appearance of “bright spot” features, suggesting that large numbers of channels were being grouped at specific sites. Our data provide clear evidence that increased numbers and altered distribution of Kv4.2 channels at the cell surface are primarily the result of reorganization of the actin cytoskeleton.

scholarly journals Slo2 potassium channel function depends on RNA editing-regulated expression of a SCYL1 protein

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Long-Gang Niu ◽  
Ping Liu ◽  
Zhao-Wen Wang ◽  
Bojun Chen
Keyword(s):  
Rna Editing ◽  
Single Channel ◽  
Expression System ◽  
Wild Type ◽  
Neuronal Function ◽  
Knockout Mutant ◽  
Open Probability ◽  
C Elegans ◽  
Evolutionarily Conserved ◽  
Regulated Expression

Slo2 potassium channels play important roles in neuronal function, and their mutations in humans may cause epilepsies and cognitive defects. However, it is largely unknown how Slo2 is regulated by other proteins. Here we show that the function of C. elegans Slo2 (SLO-2) depends on adr-1, a gene important to RNA editing. ADR-1 promotes SLO-2 function not by editing the transcripts of slo-2 but those of scyl-1, which encodes an orthologue of mammalian SCYL1. Transcripts of scyl-1 are greatly decreased in adr-1 mutants due to deficient RNA editing at a single adenosine in their 3’-UTR. SCYL-1 physically interacts with SLO-2 in neurons. Single-channel open probability (Po) of neuronal SLO-2 is ~50% lower in scyl-1 knockout mutant than wild type. Moreover, human Slo2.2/Slack Po is doubled by SCYL1 in a heterologous expression system. These results suggest that SCYL-1/SCYL1 is an evolutionarily conserved regulator of Slo2 channels.

scholarly journals Slo2 potassium channel function depends on a SCYL1 protein

2019 ◽  
Author(s):  
Long-Gang Niu ◽  
Ping Liu ◽  
Zhao-Wen Wang ◽  
Bojun Chen
Keyword(s):  
Rna Editing ◽  
Single Channel ◽  
Expression System ◽  
Mrna Level ◽  
Wild Type ◽  
Neuronal Function ◽  
Heterologous Expression System ◽  
Open Probability ◽  
C Elegans ◽  
Evolutionarily Conserved

AbstractSlo2 potassium channels play important roles in neuronal function, and their mutations in humans cause epilepsies and cognitive defects. However, little is known how Slo2 function is regulated by other proteins. Here we found that the function of C. elegans Slo2 (SLO-2) depends on adr-1, a gene important to RNA editing. However, slo-2 transcripts have no detectable RNA editing events and exhibit similar expression levels in wild type and adr-1 mutants. In contrast, mRNA level of scyl-1, which encodes an orthologue of mammalian SCYL1, is greatly reduced in adr-1 mutants due to deficient RNA editing at a single adenosine in its 3’-UTR. SCYL-1 physically interacts with SLO-2 in neurons. Single-channel open probability of SLO-2 in neurons is reduced by ∼50% in scyl-1 knockout whereas that of human Slo2.2/Slack is doubled by SCYL1 in a heterologous expression system. These results suggest that SCYL-1/SCYL1 is an evolutionarily conserved regulator of Slo2 channels.

scholarly journals Membrane-delimited Inhibition of Maxi-K Channel Activity by the Intermediate Conductance Ca2+-activated K Channel

2006 ◽  
Vol 127 (2) ◽  
pp. 159-169 ◽  
Author(s):  
Jill Thompson ◽  
Ted Begenisich
Keyword(s):  
Single Channel ◽  
Expression System ◽  
Chemical Activation ◽  
K Channel ◽  
Channel Activity ◽  
Single Family ◽  
Open Probability ◽  
K Channels ◽  
Channel Proteins ◽  
K Activity

The complexity of mammalian physiology requires a diverse array of ion channel proteins. This diversity extends even to a single family of channels. For example, the family of Ca2+-activated K channels contains three structural subfamilies characterized by small, intermediate, and large single channel conductances. Many cells and tissues, including neurons, vascular smooth muscle, endothelial cells, macrophages, and salivary glands express more than a single class of these channels, raising questions about their specific physiological roles. We demonstrate here a novel interaction between two types of Ca2+-activated K channels: maxi-K channels, encoded by the KCa1.1 gene, and IK1 channels (KCa3.1). In both native parotid acinar cells and in a heterologous expression system, activation of IK1 channels inhibits maxi-K activity. This interaction was independent of the mode of activation of the IK1 channels: direct application of Ca2+, muscarinic receptor stimulation, or by direct chemical activation of the IK1 channels. The IK1-induced inhibition of maxi-K activity occurred in small, cell-free membrane patches and was due to a reduction in the maxi-K channel open probability and not to a change in the single channel current level. These data suggest that IK1 channels inhibit maxi-K channel activity via a direct, membrane-delimited interaction between the channel proteins. A quantitative analysis indicates that each maxi-K channel may be surrounded by four IK1 channels and will be inhibited if any one of these IK1 channels opens. This novel, regulated inhibition of maxi-K channels by activation of IK1 adds to the complexity of the properties of these Ca2+-activated K channels and likely contributes to the diversity of their functional roles.

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.

Regulation of a cloned epithelial Na+ channel by its β- and γ-subunits

1997 ◽  
Vol 273 (6) ◽  
pp. C1889-C1899 ◽  
Author(s):  
Mouhamed S. Awayda ◽  
Albert Tousson ◽  
Dale J. Benos
Keyword(s):  
Fluorescence Intensity ◽  
Expression System ◽  
Fold Increase ◽  
Na Channel ◽  
Β Subunit ◽  
Open Probability ◽  
Animal Pole ◽  
Α Subunit ◽  
Amino Acid Peptide ◽  
Xenopus Oocyte Expression

Using the Xenopus oocyte expression system, we examined the mechanisms by which the β- and γ-subunits of an epithelial Na+channel (ENaC) regulate α-subunit channel activity and the mechanisms by which β-subunit truncations cause ENaC activation. Expression of α-ENaC alone produced small amiloride-sensitive currents (−43 ± 10 nA, n = 7). These currents increased >30-fold with the coexpression of β- and γ-ENaC to −1,476 ± 254 nA ( n = 20). This increase was accompanied by a 3.1- and 2.7-fold increase of membrane fluorescence intensity in the animal and vegetal poles of the oocyte, respectively, with use of an antibody directed against the α-subunit of ENaC. Truncation of the last 75 amino acids of the β-subunit COOH terminus, as found in the original pedigree of individuals with Liddle’s syndrome, caused a 4.4-fold ( n = 17) increase of the amiloride-sensitive currents compared with wild-type αβγ-ENaC. This was accompanied by a 35% increase of animal pole membrane fluorescence intensity. Injection of a 30-amino acid peptide with sequence identity to the COOH terminus of the human β-ENaC significantly reduced the amiloride-sensitive currents by 40–50%. These observations suggest a tonic inhibitory role on the channel’s open probability ( P o) by the COOH terminus of β-ENaC. We conclude that the changes of current observed with coexpression of the β- and γ-subunits or those observed with β-subunit truncation are likely the result of changes of channel density in combination with large changes of P o.

scholarly journals Therapeutic inhibition of keratinocyte TRPV3 sensory channel by local anesthetic dyclonine

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Qiang Liu ◽  
Jin Wang ◽  
Xin Wei ◽  
Juan Hu ◽  
Conghui Ping ◽  
...  
Keyword(s):  
Transient Receptor Potential ◽  
Single Channel ◽  
Receptor Potential ◽  
Epidermal Keratinocytes ◽  
Transient Receptor Potential Vanilloid ◽  
Growth Disorders ◽  
Open Probability ◽  
Gain Of Function ◽  
Sensory Channel

The multimodal sensory channel transient receptor potential vanilloid-3 (TRPV3) is expressed in epidermal keratinocytes and implicated in chronic pruritus, allergy, and inflammation-related skin disorders. Gain-of-function mutations of TRPV3 cause hair growth disorders in mice and Olmsted Syndrome in human. We here report that mouse and human TRPV3 channel is targeted by the clinical medication dyclonine that exerts a potent inhibitory effect. Accordingly, dyclonine rescued cell death caused by gain-of-function TRPV3 mutations and suppressed pruritus symptoms in vivo in mouse model. At the single-channel level, dyclonine inhibited TRPV3 open probability but not the unitary conductance. By molecular simulations and mutagenesis, we further uncovered key residues in TRPV3 pore region that could toggle the inhibitory efficiency of dyclonine. The functional and mechanistic insights obtained on dyclonine-TRPV3 interaction will help to conceive updated therapeutics for skin inflammation.

scholarly journals Dual Effect of Tamoxifen on Arterial KCa Channels Does Not Depend on the Presence of the β1 Subunit

2005 ◽  
Vol 280 (23) ◽  
pp. 21739-21747 ◽  
Author(s):  
Guillermo J. Pérez
Keyword(s):  
Molecular Mechanisms ◽  
Single Channel ◽  
Protective Role ◽  
Wild Type ◽  
Open Probability ◽  
Dual Effect ◽  
Knock Out ◽  
Kca Channels ◽  
Molecular Specificity ◽  
Excised Patches

Tamoxifen has been reported to directly activate large conductance calcium-activated potassium (KCa) channels through the KCa β1 subunit, suggesting a cardio-protective role of this compound. The present study using knock-out (KO) mice for the KCa channel β1 subunit was aimed at understanding the molecular mechanisms of the effects of tamoxifen on arterial smooth muscle KCa channels. Single channel studies were conducted in excised patches from cerebral artery myocytes from both wild-type and KO animals. The present data demonstrated that tamoxifen can inhibit arterial KCa channels due to a major decrease in channel open probability (Po), a mechanism different from the reduction in single channel amplitude reported previously and also observed in the present work. A tamoxifen-induced decrease in Po was present in arterial KCa channels from both wild-type and β1 KO animals. This inhibition was concentration-dependent and partially reversible with a half-maximal concentration constant IC50 of 2.6 μm. The effect of tamoxifen was actually dual Single channel kinetic analysis showed that tamoxifen shortens both mean closed time and mean open time; the latter is probably due to an intermediate duration voltage-independent blocking mechanism. Thus, tamoxifen block would predominate when KCa channel Po is >0.1–0.2, limiting the maximum Po, whereas a leftward shift in voltage or Ca2+ activation curves can be observed for Po values lower than those values. This dual effect of tamoxifen appears to be independent of the β1 subunit. The molecular specificity of tamoxifen, or eventually other xenoestrogen derivatives, for the KCa channel β1 subunit is uncertain.

scholarly journals A molecular link between activation and inactivation of sodium channels.

1995 ◽  
Vol 106 (4) ◽  
pp. 641-658 ◽  
Author(s):  
M E O'Leary ◽  
L Q Chen ◽  
R G Kallen ◽  
R Horn
Keyword(s):  
Sodium Channels ◽  
Voltage Dependence ◽  
Single Channel ◽  
Critical Role ◽  
Channel Measurements ◽  
Wild Type ◽  
Open Probability ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Normal Coupling

A pair of tyrosine residues, located on the cytoplasmic linker between the third and fourth domains of human heart sodium channels, plays a critical role in the kinetics and voltage dependence of inactivation. Substitution of these residues by glutamine (Y1494Y1495/QQ), but not phenylalanine, nearly eliminates the voltage dependence of the inactivation time constant measured from the decay of macroscopic current after a depolarization. The voltage dependence of steady state inactivation and recovery from inactivation is also decreased in YY/QQ channels. A characteristic feature of the coupling between activation and inactivation in sodium channels is a delay in development of inactivation after a depolarization. Such a delay is seen in wild-type but is abbreviated in YY/QQ channels at -30 mV. The macroscopic kinetics of activation are faster and less voltage dependent in the mutant at voltages more negative than -20 mV. Deactivation kinetics, by contrast, are not significantly different between mutant and wild-type channels at voltages more negative than -70 mV. Single-channel measurements show that the latencies for a channel to open after a depolarization are shorter and less voltage dependent in YY/QQ than in wild-type channels; however the peak open probability is not significantly affected in YY/QQ channels. These data demonstrate that rate constants involved in both activation and inactivation are altered in YY/QQ channels. These tyrosines are required for a normal coupling between activation voltage sensors and the inactivation gate. This coupling insures that the macroscopic inactivation rate is slow at negative voltages and accelerated at more positive voltages. Disruption of the coupling in YY/QQ alters the microscopic rates of both activation and inactivation.

A conserved cytoplasmic region of ROMK modulates pH sensitivity, conductance, and gating

1997 ◽  
Vol 273 (4) ◽  
pp. F516-F529 ◽  
Author(s):  
Han Choe ◽  
Hao Zhou ◽  
Lawrence G. Palmer ◽  
Henry Sackin
Keyword(s):  
Single Channel ◽  
Ph Sensitivity ◽  
Channel Noise ◽  
Wild Type ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Hydrophobic Region ◽  
K Secretion ◽  
Single Channel Currents

ROMK channels play a key role in overall K balance by controlling K secretion across the apical membrane of mammalian cortical collecting tubule. In contrast to the family of strong inward rectifiers (IRKs), ROMK channels are markedly sensitive to intracellular pH. Using Xenopus oocytes, we have confirmed this pH sensitivity at both the single-channel and whole cell level. Reduction of oocyte pH from 6.8 to 6.4 (using a permeant acetate buffer) reduced channel open probability from 0.76 ± 0.02 to near zero ( n = 8), without altering single-channel conductance. This was due to the appearance of a long-lived closed state at low internal pH. We have confirmed that a lysine residue (K61 on ROMK2; K80 on ROMK1), NH2 terminal to the first putative transmembrane segment (M1), is primarily responsible for conferring a steep pH sensitivity to ROMK (B. Fakler, J. Schultz, J. Yang, U. Schulte, U. Bråandle, H. P. Zenner, L. Y. Jan, and J. P. Ruppersberg. EMBO J. 15: 4093–4099, 1996). However, the apparent p K a of ROMK also depends on another residue in a highly conserved, mildly hydrophobic area: T51 on ROMK2 (T70 on ROMK1). Replacing this neutral threonine (T51) with a negatively charged glutamate shifted the apparent p K a for inward conductance from 6.5 ± 0.01 ( n = 8, wild type) to 7.0 ± 0.02 ( n = 5, T51E). On the other hand, replacing T51 with a positively charged lysine shifted the apparent p K a in the opposite direction, from 6.5 ± 0.01 ( n = 8, wild type) to 6.0 ± 0.02 ( n = 9, T51K). The opposite effects of the glutamate and lysine substitutions at position 51 (ROMK2) are consistent with a model in which T51 is physically close to K61 and alters either the local pH or the apparent p K a via an electrostatic mechanism. In addition to its effects on pH sensitivity, the mutation T51E also decreased single-channel conductance from 34.0 ± 1.0 pS ( n = 8, wild type) to 17.4 ± 1 pS ( n = 9, T51E), reversed the voltage gating of the channel, and significantly increased open-channel noise. These effects on single-channel currents suggest that the T51 residue, located in a mildly hydrophobic area of ROMK2, also interacts with the hydrophobic region of the permeation pathway.

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