scholarly journals Theoretical Investigation of the Mechanism by which A Gain-of-Function Mutation of the TRPM4 Channel Causes Conduction Block

2021 ◽  
Vol 22 (16) ◽  
pp. 8513
Author(s):  
Yaopeng Hu ◽  
Qin Li ◽  
Yanghua Shen ◽  
Takayuki Fujita ◽  
Xin Zhu ◽  
...  
Keyword(s):  
Single Channel ◽  
Conduction Block ◽  
Maximal Activity ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Resting Membrane Potential ◽  
Single Mutation ◽  
Wild Type ◽  
Mutant Channel ◽  
Trpm4 Channel

In the heart, TRPM4 is most abundantly distributed in the conduction system. Previously, a single mutation, ‘E7K’, was identified in its distal N-terminus to cause conduction disorder because of enhanced cell-surface expression. It remains, however, unclear how this expression increase leads to conduction failure rather than abnormally enhanced cardiac excitability. To address this issue theoretically, we mathematically formulated the gating kinetics of the E7K-mutant TRPM4 channel by a combined use of voltage jump analysis and ionomycin-perforated cell-attached recording technique and incorporated the resultant rate constants of opening and closing into a human Purkinje fiber single-cell action potential (AP) model (Trovato model) to perform 1D-cable simulations. The results from TRPM4 expressing HEK293 cells showed that as compared with the wild-type, the open state is much preferred in the E7K mutant with increased voltage-and Ca2+-sensitivities. These theoretical predictions were confirmed by power spectrum and single channel analyses of expressed wild-type and E7K-mutant TRPM4 channels. In our modified Trovato model, the facilitated opening of the E7K mutant channel markedly prolonged AP duration with concomitant depolarizing shifts of the resting membrane potential in a manner dependent on the channel density (or maximal activity). This was, however, little evident in the wild-type TRPM4 channel. Moreover, 1D-cable simulations with the modified Trovato model revealed that increasing the density of E7K (but not of wild-type) TRPM4 channels progressively reduced AP conduction velocity eventually culminating in complete conduction block. These results clearly suggest the brady-arrhythmogenicity of the E7K mutant channel which likely results from its pathologically enhanced activity.

scholarly journals Serine 129 phosphorylation of membrane-associated α-synuclein modulates dopamine transporter function in a G protein–coupled receptor kinase–dependent manner

2013 ◽  
Vol 24 (11) ◽  
pp. 1649-1660 ◽  
Author(s):  
Susumu Hara ◽  
Shigeki Arawaka ◽  
Hiroyasu Sato ◽  
Youhei Machiya ◽  
Can Cui ◽  
...  
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Dopamine Transporter ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Receptor Kinase ◽  
Wild Type ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

Most α-synuclein (α-syn) deposited in Lewy bodies, the pathological hallmark of Parkinson disease (PD), is phosphorylated at Ser-129. However, the physiological and pathological roles of this modification are unclear. Here we investigate the effects of Ser-129 phosphorylation on dopamine (DA) uptake in dopaminergic SH-SY5Y cells expressing α-syn. Subcellular fractionation of small interfering RNA (siRNA)–treated cells shows that G protein–coupled receptor kinase 3 (GRK3), GRK5, GRK6, and casein kinase 2 (CK2) contribute to Ser-129 phosphorylation of membrane-associated α-syn, whereas cytosolic α-syn is phosphorylated exclusively by CK2. Expression of wild-type α-syn increases DA uptake, and this effect is diminished by introducing the S129A mutation into α-syn. However, wild-type and S129A α-syn equally increase the cell surface expression of dopamine transporter (DAT) in SH-SY5Y cells and nonneuronal HEK293 cells. In addition, siRNA-mediated knockdown of GRK5 or GRK6 significantly attenuates DA uptake without altering DAT cell surface expression, whereas knockdown of CK2 has no effect on uptake. Taken together, our results demonstrate that membrane-associated α-syn enhances DA uptake capacity of DAT by GRKs-mediated Ser-129 phosphorylation, suggesting that α-syn modulates intracellular DA levels with no functional redundancy in Ser-129 phosphorylation between GRKs and CK2.

scholarly journals Molecular Basis of Ca2+ Activation of the Mouse Cardiac Ca2+ Release Channel (Ryanodine Receptor)

2001 ◽  
Vol 118 (1) ◽  
pp. 33-44 ◽  
Author(s):  
Pin Li ◽  
S.R. Wayne Chen
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Molecular Basis ◽  
Single Channel ◽  
Single Point ◽  
Hek293 Cells ◽  
Single Point Mutation ◽  
Single Channels ◽  
Wild Type ◽  
Structural And Functional Properties

Activation of the cardiac ryanodine receptor (RyR2) by Ca2+ is an essential step in excitation-contraction coupling in heart muscle. However, little is known about the molecular basis of activation of RyR2 by Ca2+. In this study, we investigated the role in Ca2+ sensing of the conserved glutamate 3987 located in the predicted transmembrane segment M2 of the mouse RyR2. Single point mutation of this conserved glutamate to alanine (E3987A) reduced markedly the sensitivity of the channel to activation by Ca2+, as measured by using single-channel recordings in planar lipid bilayers and by [3H]ryanodine binding assay. However, this mutation did not alter the affinity of [3H]ryanodine binding and the single-channel conductance. In addition, the E3987A mutant channel was activated by caffeine and ATP, was inhibited by Mg2+, and was modified by ryanodine in a fashion similar to that of the wild-type channel. Coexpression of the wild-type and mutant E3987A RyR2 proteins in HEK293 cells produced individual single channels with intermediate sensitivities to activating Ca2+. These results are consistent with the view that glutamate 3987 is a major determinant of Ca2+ sensitivity to activation of the mouse RyR2 channel, and that Ca2+ sensing by RyR2 involves the cooperative action between ryanodine receptor monomers. The results of this study also provide initial insights into the structural and functional properties of the mouse RyR2, which should be useful for studying RyR2 function and regulation in genetically modified mouse models.

scholarly journals ML277 regulates KCNQ1 single-channel amplitudes and kinetics, modified by voltage sensor state

2021 ◽  
Vol 153 (12) ◽  
Author(s):  
Jodene Eldstrom ◽  
Donald A. McAfee ◽  
Ying Dou ◽  
Yundi Wang ◽  
David Fedida
Keyword(s):  
Single Channel ◽  
Therapeutic Potential ◽  
Induced Pluripotent Stem Cell ◽  
Voltage Sensor ◽  
K Channel ◽  
Cardiac Repolarization ◽  
Wild Type ◽  
Mutant Channel ◽  
Induced Pluripotent ◽  
Sensor State

KCNQ1 is a pore-forming K+ channel subunit critically important to cardiac repolarization at high heart rates. (2R)-N-[4-(4-methoxyphenyl)-2-thiazolyl]-1-[(4-methylphenyl)sulfonyl]-2 piperidinecarboxamide, or ML277, is an activator of this channel that rescues function of pathophysiologically important mutant channel complexes in human induced pluripotent stem cell–derived cardiomyocytes, and that therefore may have therapeutic potential. Here we extend our understanding of ML277 actions through cell-attached single-channel recordings of wild-type and mutant KCNQ1 channels with voltage sensor domains fixed in resting, intermediate, and activated states. ML277 has profound effects on KCNQ1 single-channel kinetics, eliminating the flickering nature of the openings, converting them to discrete opening bursts, and increasing their amplitudes approximately threefold. KCNQ1 single-channel behavior after ML277 treatment most resembles IO state-locked channels (E160R/R231E) rather than AO state channels (E160R/R237E), suggesting that at least during ML277 treatment, KCNQ1 does not frequently visit the AO state. Introduction of KCNE1 subunits reduces the effectiveness of ML277, but some enhancement of single-channel openings is still observed.

Genistein potentiates wild-type and delta F508-CFTR channel activity

1997 ◽  
Vol 273 (3) ◽  
pp. C988-C998 ◽  
Author(s):  
T. C. Hwang ◽  
F. Wang ◽  
I. C. Yang ◽  
W. W. Reenstra
Keyword(s):  
Single Channel ◽  
Maximal Activity ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Open Time ◽  
Excised Patches ◽  
Cftr Protein ◽  
Nih 3T3 ◽  
Camp Stimulation

Effects of genistein on wild-type (wt) and delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) were studied in NIH/3T3 cells stably transfected with wt or mutant CFTR cDNA. As measured by I- efflux, half-maximal concentration of agonist (K1/2) for forskolin-dependent activation was greater for delta F508-CFTR than wt-CFTR. Genistein decreased the K1/2 for both forms of the channel and increased the maximal activity of delta F508-CFTR by 3.7-fold. In cell-attached patches, 10 microM forskolin induced minimal delta F508-CFTR activity with characteristic prolonged closed times (estimated time constant, > 30 s). Genistein increased the forskolin-induced macroscopic currents of wt-CFTR and delta F508-CFTR by 3- and 19-fold, respectively. Variance analysis suggested that in the presence of forskolin and genistein the open probabilities (Po) of wt- and delta F508-CFTR were identical. In single-channel studies, at maximal adenosine 3',5'-cyclic monophosphate (cAMP) stimulation, genistein increased the Po of wt-CFTR by prolonging the open time, but, at submaximal cAMP stimulation, the Po was increased by prolonging the open time and shortening the closed time. In excised patches with CFTR channels preactivated in the cell-attached mode, genistein increased ATP-dependent wt- and delta F508-CFTR current about twofold by prolonging the open time. Our results thus suggest that phosphorylation-dependent activation of delta F508-CFTR is defective and that genistein corrects this defect at least in part by binding to the CFTR protein.

scholarly journals SAP97 regulates Kir2.3 channels by multiple mechanisms

2009 ◽  
Vol 297 (4) ◽  
pp. H1387-H1397 ◽  
Author(s):  
Karen L. Vikstrom ◽  
Ravi Vaidyanathan ◽  
Susan Levinsohn ◽  
Ryan P. O'Connell ◽  
Yueming Qian ◽  
...  
Keyword(s):  
Cell Surface ◽  
Single Channel ◽  
Hek293 Cells ◽  
Scaffolding Protein ◽  
Cell Surface Expression ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Whole Cell ◽  
The Impact

We examined the impact of coexpressing the inwardly rectifying potassium channel, Kir2.3, with the scaffolding protein, synapse-associated protein (SAP) 97, and determined that coexpression of these proteins caused an approximately twofold increase in current density. A combination of techniques was used to determine if the SAP97-induced increase in Kir2.3 whole cell currents resulted from changes in the number of channels in the cell membrane, unitary channel conductance, or channel open probability. In the absence of SAP97, Kir2.3 was found predominantly in a cytoplasmic, vesicular compartment with relatively little Kir2.3 localized to the plasma membrane. The introduction of SAP97 caused a redistribution of Kir2.3, leading to prominent colocalization of Kir2.3 and SAP97 and a modest increase in cell surface Kir2.3. The median Kir2.3 single channel conductance in the absence of SAP97 was ∼13 pS, whereas coexpression of SAP97 led to a wide distribution of channel events with three distinct peaks centered at 16, 29, and 42 pS. These changes occurred without altering channel open probability, current rectification properties, or pH sensitivity. Thus association of Kir2.3 with SAP97 in HEK293 cells increased channel cell surface expression and unitary channel conductance. However, changes in single channel conductance play the major role in determining whole cell currents in this model system. We further suggest that the SAP97 effect results from SAP97 binding to the Kir2.3 COOH-terminal domain and altering channel conformation.

scholarly journals A Point Mutation in the Human Melanin Concentrating Hormone Receptor 1 Reveals an Important Domain for Cellular Trafficking

2005 ◽  
Vol 19 (10) ◽  
pp. 2579-2590 ◽  
Author(s):  
Jun Fan ◽  
Stephen J. Perry ◽  
Yinghong Gao ◽  
David A. Schwarz ◽  
Richard A. Maki
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Point Mutation ◽  
Hormone Receptor ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Single Mutation ◽  
Wild Type ◽  
Melanin Concentrating Hormone ◽  
Type Receptor

Abstract G protein-coupled receptors (GPCRs) are heptahelical integral membrane proteins that require cell surface expression to elicit their effects. The lack of appropriate expression of GPCRs may be the underlying cause of a number of inherited disorders. There is evidence that newly synthesized GPCRs must attain a specific conformation for their correct trafficking to the cell surface. In this study, we show that a single point mutation in human melanin-concentrating hormone receptor (hMCHR1) at position 255 (T255A), which is located at the junction of intracellular loop 3 and transmembrane domain 6, reduces the hMCHR1 cell surface expression level to 20% of that observed for the wild-type receptor. Most of these mutant receptors are located intracellularly, as opposed to the wild-type receptor, which is located primarily on the cell surface. Immunoprecipitation experiments show that hMCHR1-T255A has reduced glycosylation compared with the wild-type receptor and is associated with the chaperone protein, calnexin, and it colocalizes in the endoplasmic reticulum with KDEL-containing proteins. We also demonstrate that a cell-permeable small molecule antagonist of hMCHR1 can function as a pharmacological chaperone to restore cell surface expression of this and other MCHR1 mutants to wild-type levels. Once rescued, the T255A mutant couples to Gq proteins as efficiently as the wild-type receptor. These data suggest that this single mutation produces an hMCHR1 that folds incorrectly, resulting in its retention in the endoplasmic reticulum, but once rescued to the cell surface can still function normally.

scholarly journals WNK4 inhibition of ENaC is independent of Nedd4-2-mediated ENaC ubiquitination

2013 ◽  
Vol 305 (1) ◽  
pp. F31-F41 ◽  
Author(s):  
Ling Yu ◽  
Hui Cai ◽  
Qian Yue ◽  
Abdel A. Alli ◽  
DeXuan Wang ◽  
...  
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Hek293 Cells ◽  
Wild Type ◽  
Distal Nephron ◽  
Epithelial Sodium Channels ◽  
Open Probability ◽  
Western Blots ◽  
Current Increase ◽  
Rate Of Increase

A serine-threonine protein kinase, WNK4, reduces Na+ reabsorption and K+ secretion in the distal convoluted tubule by reducing trafficking of the thiazide-sensitive Na-Cl cotransporter to and enhancing renal outer medullary potassium channel retrieval from the apical membrane. Epithelial sodium channels (ENaC) in the distal nephron also play a role in regulating Na+ reabsorption and are also regulated by WNK4, but the mechanism is unclear. In A6 distal nephron cells, transepithelial current measurement and single channel recording show that WNK4 inhibits ENaC activity. Analysis of the number of channel per patch shows that WNK4 reduces channel number but has no effect on channel open probability. Western blots of apical and total ENaC provide additional evidence that WNK4 reduces apical as well as total ENaC expression. WNK4 enhances ENaC internalization independent of Nedd4-2-mediated ENaC ubiquitination. WNK4 also reduced the amount of ENaC available for recycling but has no effect on the rate of transepithelial current increase to forskolin. In contrast, Nedd4-2 not only reduced ENaC in the recycling pool but also decreased the rate of increase of current after forskolin. WNK4 associates with wild-type as well as Liddle's mutated ENaC, and WNK4 reduces both wild-type and mutated ENaC expressed in HEK293 cells.

A novel I551F variant of Na+/HCO3- cotransporter NBCe1-A shows reduced cell surface expression, resulting in diminished transport activity

2021 ◽  
Author(s):  
Osamu Yamazaki ◽  
Maho Yamashita ◽  
Jinping Li ◽  
Fumika Ochiai-Homma ◽  
Tadashi Yoshida ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Surface Expression ◽  
Dominant Negative ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Dominant Negative Effect ◽  
Transport Activity ◽  
Wild Type ◽  
Single Nucleotide Variants

Homozygous mutations in SLC4A4, encoding the electrogenic Na+/HCO3- cotransporter NBCe1, cause proximal renal tubular acidosis (pRTA) associated with extrarenal symptoms. Although 17 mutated sites in SLC4A4 have thus far been identified among pRTA patients, physiological significance of other nonsynonymous single nucleotide variants (SNVs) remains largely undetermined. Here, we investigated the functional properties of SNVs in NBCe1. From NCBI dbSNP database, we identified 13 SNVs that have not previously been characterized in highly conserved, transmembrane domains of NBCe1-A. Immunocytochemical analysis revealed that I551F variant was present predominantly in the cytoplasm in HEK293 cells, whereas all other SNVs did not show as dramatic a change in subcellular distribution. Western blot analysis in HEK293 cells demonstrated that the I551F variant showed impaired glycosylation and a 69 % reduction in cell surface levels. To determine the role of I551 in more detail, we examined the significance of various artificial mutants both in non-polarized HEK293 cells and polarized MDCK cells, which indicated that only I551F substitution resulted in cytoplasmic retention. Moreover, functional analysis using Xenopus oocytes demonstrated that the I551F variant had a significantly reduced activity corresponding to 39 % of that of wild-type, whereas any other SNVs and artificial I551 mutants did not show significant changes in activity. Finally, immunofluorescence study in HEK293 cells indicated that the I551F variant retains wild-type NBCe1-A in the cytoplasm. These data demonstrate that I551F-NBCe1-A shows impaired transport activity predominantly through cytoplasmic retention, and suggest that the variant can have a dominant-negative effect by forming complexes with wild-type NBCe1-A.

Molecular and genetic characterization of natural variants of HIV-1 Nef gene from North India and its functional implication in down-regulation of MHC-I and CD-4

2020 ◽  
Vol 18 ◽  
Author(s):  
J. Singh ◽  
L. Ronsard ◽  
M. Pandey ◽  
R. Kapoor ◽  
V.G. Ramachandran ◽  
...  
Keyword(s):  
Biological Activities ◽  
Sequence Similarity ◽  
Eukaryotic Expression ◽  
North India ◽  
Cell Surface Expression ◽  
Functional Domains ◽  
Wild Type ◽  
Down Regulation ◽  
Subtype B ◽  
Hiv 1

Background: HIV-1 Nef is an important accessory protein with multiple effector functions. Genetic studies of HIV-1 Nef gene shows extensive genetic diversity and the functional studies have been carried out mostly with Nef derived from regions dominated by subtype B (North America & Europe). Objective: This study was carried out to characterize genetic variations of the Nef gene from HIV-1 infected individuals from North-India and to find out their functional implications. Methods: The unique representative variants were sub-cloned in eukaryotic expression vector and further characterized with respect to their ability to down regulate cell surface expression of CD4 and MHC-1molecules. Results: The phylogenetic analysis of Nef variants revealed sequence similarity with either consensus subtype B or B/C recombinants. Boot scan analysis of some of our variants showed homology to B/C recombinant and some to wild type Nef B. Extensive variations were observed in most of the variants. The dN/dS ratio revealed 80% purifying selection and 20% diversifying selection implying the importance of mutations in Nef variants. Intracellular stability of Nef variants differed greatly when compared with wild type Nef B and C. There were some variants that possessed mutations in the functional domains of Nef and responsible for its differential CD4 and MHC-1 down regulation activity. Conclusion: We observed enhanced biological activities in some of the variants, perhaps arising out of amino acid substitutions in their functional domains. The CD4 and MHC-1 down-regulation activity of Nef is likely to confer immense survival advantage allowing the most rare genotype in a population to become the most abundant after a single selection event.

scholarly journals The NALCN Channel Regulator UNC-80 Functions in a Subset of Interneurons To Regulate Caenorhabditis elegans Reversal Behavior

2019 ◽  
Vol 10 (1) ◽  
pp. 199-210 ◽  
Author(s):  
Chuanman Zhou ◽  
Jintao Luo ◽  
Xiaohui He ◽  
Qian Zhou ◽  
Yunxia He ◽  
...  
Keyword(s):  
Plant Hormone ◽  
Neuronal Excitability ◽  
Resting Membrane Potential ◽  
Loss Of Function ◽  
Wild Type ◽  
C Elegans ◽  
Link Type ◽  
Complex Functions ◽  
And Function ◽  
Multiple Loss

NALCN (Na+leak channel, non-selective) is a conserved, voltage-insensitive cation channel that regulates resting membrane potential and neuronal excitability. UNC79 and UNC80 are key regulators of the channel function. However, the behavioral effects of the channel complex are not entirely clear and the neurons in which the channel functions remain to be identified. In a forward genetic screen for C. elegans mutants with defective avoidance response to the plant hormone methyl salicylate (MeSa), we isolated multiple loss-of-function mutations in unc-80 and unc-79. C. elegans NALCN mutants exhibited similarly defective MeSa avoidance. Interestingly, NALCN, unc-80 and unc-79 mutants all showed wild type-like responses to other attractive or repelling odorants, suggesting that NALCN does not broadly affect odor detection or related forward and reversal behaviors. To understand in which neurons the channel functions, we determined the identities of a subset of unc-80-expressing neurons. We found that unc-79 and unc-80 are expressed and function in overlapping neurons, which verified previous assumptions. Neuron-specific transgene rescue and knockdown experiments suggest that the command interneurons AVA and AVE and the anterior guidepost neuron AVG can play a sufficient role in mediating unc-80 regulation of the MeSa avoidance. Though primarily based on genetic analyses, our results further imply that MeSa might activate NALCN by direct or indirect actions. Altogether, we provide an initial look into the key neurons in which the NALCN channel complex functions and identify a novel function of the channel in regulating C. elegans reversal behavior through command interneurons.

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