scholarly journals Stac3 enhances expression of human CaV1.1 in Xenopus oocytes and reveals gating pore currents in HypoPP mutant channels

2018 ◽  
Vol 150 (3) ◽  
pp. 475-489 ◽  
Author(s):  
Fenfen Wu ◽  
Marbella Quinonez ◽  
Marino DiFranco ◽  
Stephen C. Cannon
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Ionic Currents ◽  
Periodic Paralysis ◽  
Xenopus Laevis Oocytes ◽  
Missense Mutations ◽  
Membrane Expression ◽  
Functional Studies ◽  
Gating Charge ◽  
Arginine Residues

Mutations of CaV1.1, the pore-forming subunit of the L-type Ca2+ channel in skeletal muscle, are an established cause of hypokalemic periodic paralysis (HypoPP). However, functional assessment of HypoPP mutant channels has been hampered by difficulties in achieving sufficient plasma membrane expression in cells that are not of muscle origin. In this study, we show that coexpression of Stac3 dramatically increases the expression of human CaV1.1 (plus α2-δ1b and β1a subunits) at the plasma membrane of Xenopus laevis oocytes. In voltage-clamp studies with the cut-open oocyte clamp, we observe ionic currents on the order of 1 μA and gating charge displacements of ∼0.5–1 nC. Importantly, this high expression level is sufficient to ascertain whether HypoPP mutant channels are leaky because of missense mutations at arginine residues in S4 segments of the voltage sensor domains. We show that R528H and R528G in S4 of domain II both support gating pore currents, but unlike other R/H HypoPP mutations, R528H does not conduct protons. Stac3-enhanced membrane expression of CaV1.1 in oocytes increases the throughput for functional studies of disease-associated mutations and is a new platform for investigating the voltage-dependent properties of CaV1.1 without the complexity of the transverse tubule network in skeletal muscle.

scholarly journals Disrupted coupling of gating charge displacement to Na+ current activation for DIIS4 mutations in hypokalemic periodic paralysis

2014 ◽  
Vol 144 (2) ◽  
pp. 137-145 ◽  
Author(s):  
Wentao Mi ◽  
Volodymyr Rybalchenko ◽  
Stephen C. Cannon
Keyword(s):  
Periodic Paralysis ◽  
Specific Conductance ◽  
Resting Potential ◽  
Missense Mutations ◽  
Na Current ◽  
Hypokalemic Periodic Paralysis ◽  
Gating Charge ◽  
Arginine Residues ◽  
Charge Displacement ◽  
Low K

Missense mutations at arginine residues in the S4 voltage-sensor domains of NaV1.4 are an established cause of hypokalemic periodic paralysis, an inherited disorder of skeletal muscle involving recurrent episodes of weakness in conjunction with low serum K+. Expression studies in oocytes have revealed anomalous, hyperpolarization-activated gating pore currents in mutant channels. This aberrant gating pore conductance creates a small inward current at the resting potential that is thought to contribute to susceptibility to depolarization in low K+ during attacks of weakness. A critical component of this hypothesis is the magnitude of the gating pore conductance relative to other conductances that are active at the resting potential in mammalian muscle: large enough to favor episodes of paradoxical depolarization in low K+, yet not so large as to permanently depolarize the fiber. To improve the estimate of the specific conductance for the gating pore in affected muscle, we sequentially measured Na+ current through the channel pore, gating pore current, and gating charge displacement in oocytes expressing R669H, R672G, or wild-type NaV1.4 channels. The relative conductance of the gating pore to that of the pore domain pathway for Na+ was 0.03%, which implies a specific conductance in muscle from heterozygous patients of ∼10 µS/cm2 or 1% of the total resting conductance. Unexpectedly, our data also revealed a substantial decoupling between gating charge displacement and peak Na+ current for both R669H and R672G mutant channels. This decoupling predicts a reduced Na+ current density in affected muscle, consistent with the observations that the maximal dV/dt and peak amplitude of the action potential are reduced in fibers from patients with R672G and in a knock-in mouse model of R669H. The defective coupling between gating charge displacement and channel activation identifies a previously unappreciated mechanism that contributes to the reduced excitability of affected fibers seen with these mutations and possibly with other R/X mutations of S4 of NaV, CaV, and KV channels associated with human disease.

scholarly journals Myotonia in a patient with a mutation in an S4 arginine residue associated with hypokalaemic periodic paralysis and a concomitant synonymous CLCN1 mutation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Michael G. Thor ◽  
Vinojini Vivekanandam ◽  
Marisol Sampedro-Castañeda ◽  
S. Veronica Tan ◽  
Karen Suetterlin ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Phenotypic Variability ◽  
Transmembrane Helix ◽  
Periodic Paralysis ◽  
Loss Of Function ◽  
Leak Current ◽  
Muscle Action Potential ◽  
Arginine Residues ◽  
Pore Domain ◽  
Clcn1 Mutation

AbstractThe sarcolemmal voltage gated sodium channel NaV1.4 conducts the key depolarizing current that drives the upstroke of the skeletal muscle action potential. It contains four voltage-sensing domains (VSDs) that regulate the opening of the pore domain and ensuing permeation of sodium ions. Mutations that lead to increased NaV1.4 currents are found in patients with myotonia or hyperkalaemic periodic paralysis (HyperPP). Myotonia is also caused by mutations in the CLCN1gene that result in loss-of-function of the skeletal muscle chloride channel ClC-1. Mutations affecting arginine residues in the fourth transmembrane helix (S4) of the NaV1.4 VSDs can result in a leak current through the VSD and hypokalemic periodic paralysis (HypoPP), but these have hitherto not been associated with myotonia. We report a patient with an Nav1.4 S4 arginine mutation, R222Q, presenting with severe myotonia without fulminant paralytic episodes. Other mutations affecting the same residue, R222W and R222G, have been found in patients with HypoPP. We show that R222Q channels have enhanced activation, consistent with myotonia, but also conduct a leak current. The patient carries a concomitant synonymous CLCN1 variant that likely worsens the myotonia and potentially contributes to the amelioration of muscle paralysis. Our data show phenotypic variability for different mutations affecting the same S4 arginine that have implications for clinical therapy.

scholarly journals Cooh-Terminal Truncated Alpha1S Subunits Conduct Current Better than Full-Length Dihydropyridine Receptors

2000 ◽  
Vol 116 (3) ◽  
pp. 341-348 ◽  
Author(s):  
James A. Morrill ◽  
Stephen C. Cannon
Keyword(s):  
Skeletal Muscle ◽  
Expression System ◽  
Ionic Currents ◽  
Full Length ◽  
Charge Movement ◽  
Functional Roles ◽  
Dihydropyridine Receptors ◽  
Gating Charge ◽  
Channel Opening ◽  
Voltage Gated Channels

Skeletal muscle dihydropyridine (DHP) receptors function both as voltage-activated Ca2+ channels and as voltage sensors for coupling membrane depolarization to release of Ca2+ from the sarcoplasmic reticulum. In skeletal muscle, the principal or α1S subunit occurs in full-length (∼10% of total) and post-transcriptionally truncated (∼90%) forms, which has raised the possibility that the two functional roles are subserved by DHP receptors comprised of different sized α1S subunits. We tested the functional properties of each form by injecting oocytes with cRNAs coding for full-length (α1S) or truncated (α1SΔC) α subunits. Both translation products were expressed in the membrane, as evidenced by increases in the gating charge (Qmax 80–150 pC). Thus, oocytes provide a robust expression system for the study of gating charge movement in α1S, unencumbered by contributions from other voltage-gated channels or the complexities of the transverse tubules. As in recordings from skeletal muscle, for heterologously expressed channels the peak inward Ba2+ currents were small relative to Qmax. The truncated α1SΔC protein, however, supported much larger ionic currents than the full-length product. These data raise the possibility that DHP receptors containing the more abundant, truncated form of the α1S subunit conduct the majority of the L-type Ca2+ current in skeletal muscle. Our data also suggest that the carboxyl terminus of the α1S subunit modulates the coupling between charge movement and channel opening.

scholarly journals The inhibition of monocarboxylate transporter 2 (MCT2) by AR-C155858 is modulated by the associated ancillary protein

2010 ◽  
Vol 431 (2) ◽  
pp. 217-225 ◽  
Author(s):  
Matthew J. Ovens ◽  
Christine Manoharan ◽  
Marieangela C. Wilson ◽  
Clarey M. Murray ◽  
Andrew P. Halestrap
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Monocarboxylate Transporter ◽  
Xenopus Laevis Oocytes ◽  
Transport Activity ◽  
Membrane Expression ◽  
Binding Partner ◽  
Reverse Transcription Pcr ◽  
C Terminus ◽  
Plasma Membrane Expression

In mammalian cells, MCTs (monocarboxylate transporters) require association with an ancillary protein to enable plasma membrane expression of the active transporter. Basigin is the preferred binding partner for MCT1, MCT3 and MCT4, and embigin for MCT2. In rat and rabbit erythrocytes, MCT1 is associated with embigin and basigin respectively, but its sensitivity to inhibition by AR-C155858 was found to be identical. Using RT (reverse transcription)–PCR, we have shown that Xenopus laevis oocytes contain endogenous basigin, but not embigin. Co-expression of exogenous embigin was without effect on either the expression of MCT1 or its inhibition by AR-C155858. In contrast, expression of active MCT2 at the plasma membrane of oocytes was significantly enhanced by co-expression of exogenous embigin. This additional transport activity was insensitive to inhibition by AR-C155858 unlike that by MCT2 expressed with endogenous basigin that was potently inhibited by AR-C155858. Chimaeras and C-terminal truncations of MCT1 and MCT2 were also expressed in oocytes in the presence and absence of exogenous embigin. L-Lactate Km values for these constructs were determined and revealed that the TM (transmembrane) domains of an MCT, most probably TM7–TM12, but not the C-terminus, are the major determinants of L-lactate affinity, whereas the associated ancillary protein has little or no effect. Inhibitor titrations of lactate transport by these constructs indicated that embigin modulates MCT2 sensitivity to AR-C155858 through interactions with both the intracellular C-terminus and TMs 3 and 6 of MCT2. The C-terminus of MCT2 was found to be essential for its expression with endogenous basigin.

Hyperkalemic periodic paralysis M1592V mutation modifies activation in human skeletal muscle Na+ channel

1999 ◽  
Vol 276 (1) ◽  
pp. C259-C266 ◽  
Author(s):  
Cecilia V. Rojas ◽  
Alan Neely ◽  
Gabriela Velasco-Loyden ◽  
Verónica Palma ◽  
Manuel Kukuljan
Keyword(s):  
Skeletal Muscle ◽  
Human Skeletal Muscle ◽  
Periodic Paralysis ◽  
Na Channel ◽  
Xenopus Laevis Oocytes ◽  
Functional Difference ◽  
Wild Type ◽  
Hyperkalemic Periodic Paralysis ◽  
Activation Curve ◽  
Inactivation Curve

Mutations in the human skeletal muscle Na+ channel underlie the autosomal dominant disease hyperkalemic periodic paralysis (HPP). Muscle fibers from affected individuals exhibit sustained Na+ currents thought to depolarize the sarcolemma and thus inactivate normal Na+ channels. We expressed human wild-type or M1592V mutant α-subunits with the β1-subunit in Xenopus laevis oocytes and recorded Na+ currents using two-electrode and cut-open oocyte voltage-clamp techniques. The most prominent functional difference between M1592V mutant and wild-type channels is a 5- to 10-mV shift in the hyperpolarized direction of the steady-state activation curve. The shift in the activation curve for the mutant results in a larger overlap with the inactivation curve than that observed for wild-type channels. Accordingly, the current through M1592V channels displays a larger noninactivating component than does that through wild-type channels at membrane potentials near −40 mV. The functional properties of the M1592V mutant resemble those of the previously characterized HPP T704M mutant. Both clinically similar phenotypes arise from mutations located at a distance from the putative voltage sensor of the channel.

scholarly journals Probing biophysical sequence constraints within the transmembrane domains of rhodopsin by deep mutational scanning

2020 ◽  
Vol 6 (10) ◽  
pp. eaay7505 ◽  
Author(s):  
Wesley D. Penn ◽  
Andrew G. McKee ◽  
Charles P. Kuntz ◽  
Hope Woods ◽  
Veronica Nash ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Evolutionary Analysis ◽  
Missense Mutations ◽  
Evolutionary Sequence ◽  
Membrane Expression ◽  
Plasma Membrane Expression ◽  
Tm Domain ◽  
And Function ◽  
Sequence Constraints

Membrane proteins must balance the sequence constraints associated with folding and function against the hydrophobicity required for solvation within the bilayer. We recently found the expression and maturation of rhodopsin are limited by the hydrophobicity of its seventh transmembrane domain (TM7), which contains polar residues that are essential for function. On the basis of these observations, we hypothesized that rhodopsin’s expression should be less tolerant of mutations in TM7 relative to those within hydrophobic TM domains. To test this hypothesis, we used deep mutational scanning to compare the effects of 808 missense mutations on the plasma membrane expression of rhodopsin in HEK293T cells. Our results confirm that a higher proportion of mutations within TM7 (37%) decrease rhodopsin’s plasma membrane expression relative to those within a hydrophobic TM domain (TM2, 25%). These results in conjunction with an evolutionary analysis suggest solvation energetics likely restricts the evolutionary sequence space of polar TM domains.

scholarly journals A Na+ Channel Mutation Linked to Hypokalemic Periodic Paralysis Exposes a Proton-selective Gating Pore

2007 ◽  
Vol 130 (1) ◽  
pp. 11-20 ◽  
Author(s):  
Arie F. Struyk ◽  
Stephen C. Cannon
Keyword(s):  
Skeletal Muscle ◽  
Periodic Paralysis ◽  
Proton Leak ◽  
K Channel ◽  
Na Channel ◽  
Missense Mutations ◽  
Ph Homeostasis ◽  
Hypokalemic Periodic Paralysis ◽  
Voltage Sensing ◽  
Voltage Sensing Domain

The heritable muscle disorder hypokalemic periodic paralysis (HypoPP) is characterized by attacks of flaccid weakness, brought on by sustained sarcolemmal depolarization. HypoPP is genetically linked to missense mutations at charged residues in the S4 voltage-sensing segments of either CaV1.1 (the skeletal muscle L-type Ca2+ channel) or NaV1.4 (the skeletal muscle voltage-gated Na+ channel). Although these mutations alter the gating of both channels, these functional defects have proven insufficient to explain the sarcolemmal depolarization in affected muscle. Recent insight into the topology of the S4 voltage-sensing domain has aroused interest in an alternative pathomechanism, wherein HypoPP mutations might generate an aberrant ionic leak conductance by unblocking the putative aqueous crevice (“gating-pore”) in which the S4 segment resides. We tested the rat isoform of NaV1.4 harboring the HypoPP mutation R663H (human R669H ortholog) at the outermost arginine of S4 in domain II for a gating-pore conductance. We found that the mutation R663H permits transmembrane permeation of protons, but not larger cations, similar to the conductance displayed by histidine substitution at Shaker K+ channel S4 sites. These results are consistent with the notion that the outermost charged residue in the DIIS4 segment is simultaneously accessible to the cytoplasmic and extracellular spaces when the voltage sensor is positioned inwardly. The predicted magnitude of this proton leak in mature skeletal muscle is small relative to the resting K+ and Cl− conductances, and is thus not likely to fully account for the aberrant sarcolemmal depolarization underlying the paralytic attacks. Rather, it is possible that a sustained proton leak may contribute to instability of VREST indirectly, for instance, by interfering with intracellular pH homeostasis.

scholarly journals Gating pore currents occur in CaV1.1 domain III mutants associated with HypoPP

2021 ◽  
Vol 153 (11) ◽  
Author(s):  
Fenfen Wu ◽  
Marbella Quinonez ◽  
Stephen C. Cannon
Keyword(s):  
Periodic Paralysis ◽  
Resting Potential ◽  
Missense Mutations ◽  
Transmembrane Segments ◽  
Conduction Pathway ◽  
Low Potassium ◽  
Arginine Residues ◽  
The One ◽  
Domain Iii ◽  
Prevailing View

Mutations in the voltage sensor domain (VSD) of CaV1.1, the α1S subunit of the L-type calcium channel in skeletal muscle, are an established cause of hypokalemic periodic paralysis (HypoPP). Of the 10 reported mutations, 9 are missense substitutions of outer arginine residues (R1 or R2) in the S4 transmembrane segments of the homologous domain II, III (DIII), or IV. The prevailing view is that R/X mutations create an anomalous ion conduction pathway in the VSD, and this so-called gating pore current is the basis for paradoxical depolarization of the resting potential and weakness in low potassium for HypoPP fibers. Gating pore currents have been observed for four of the five CaV1.1 HypoPP mutant channels studied to date, the one exception being the charge-conserving R897K in R1 of DIII. We tested whether gating pore currents are detectable for the other three HypoPP CaV1.1 mutations in DIII. For the less conserved R1 mutation, R897S, gating pore currents with exceptionally large amplitude were observed, correlating with the severe clinical phenotype of these patients. At the R2 residue, gating pore currents were detected for R900G but not R900S. These findings show that gating pore currents may occur with missense mutations at R1 or R2 in S4 of DIII and that the magnitude of this anomalous inward current is mutation specific.

scholarly journals Electrogenic Na/HCO3 Cotransporter (NBCe1) Variants Expressed in Xenopus Oocytes: Functional Comparison and Roles of the Amino and Carboxy Termini

2006 ◽  
Vol 127 (6) ◽  
pp. 639-658 ◽  
Author(s):  
Suzanne D. McAlear ◽  
Xiaofen Liu ◽  
Jennifer B. Williams ◽  
Carmel M. McNicholas-Bevensee ◽  
Mark O. Bevensee
Keyword(s):  
Plasma Membrane ◽  
Voltage Clamp ◽  
Outward Current ◽  
Amino Terminus ◽  
Xenopus Laevis Oocytes ◽  
Carboxy Terminus ◽  
Membrane Expression ◽  
Whole Cell ◽  
Plasma Membrane Expression ◽  
The Mean

Using pH- and voltage-sensitive microelectrodes, as well as the two-electrode voltage-clamp and macropatch techniques, we compared the functional properties of the three NBCe1 variants (NBCe1-A, -B, and -C) with different amino and/or carboxy termini expressed in Xenopus laevis oocytes. Oocytes expressing rat brain NBCe1-B and exposed to a CO2/HCO3− solution displayed all the hallmarks of an electrogenic Na+/HCO3− cotransporter: (a) a DIDS-sensitive pHi recovery following the initial CO2-induced acidification, (b) an instantaneous hyperpolarization, and (c) an instantaneous Na+-dependent outward current under voltage-clamp conditions (−60 mV). All three variants had similar external HCO3− dependencies (apparent KM of 4–6 mM) and external Na+ dependencies (apparent KM of 21–36 mM), as well as similar voltage dependencies. However, voltage-clamped oocytes (−60 mV) expressing NBCe1-A exhibited peak HCO3−-stimulated NBC currents that were 4.3-fold larger than the currents seen in oocytes expressing the most dissimilar C variant. Larger NBCe1-A currents were also observed in current–voltage relationships. Plasma membrane expression levels as assessed by single oocyte chemiluminescence with hemagglutinin-tagged NBCs were similar for the three variants. In whole-cell experiments (Vm = −60 mV), removing the unique amino terminus of NBCe1-A reduced the mean HCO3−-induced NBC current 55%, whereas removing the different amino terminus of NBCe1-C increased the mean NBC current 2.7-fold. A similar pattern was observed in macropatch experiments. Thus, the unique amino terminus of NBCe1-A stimulates transporter activity, whereas the different amino terminus of the B and C variants inhibits activity. One or more cytosolic factors may also contribute to NBCe1 activity based on discrepancies between macropatch and whole-cell currents. While the amino termini influence transporter function, the carboxy termini influence plasma membrane expression. Removing the entire cytosolic carboxy terminus of NBCe1-C, or the different carboxy terminus of the A/B variants, causes a loss of NBC activity due to low expression at the plasma membrane.

scholarly journals The role of action potential changes in depolarization-induced failure of excitation contraction coupling in mouse skeletal muscle

eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Xueyong Wang ◽  
Murad Nawaz ◽  
Chris DuPont ◽  
Jessica H Myers ◽  
Steve RA Burke ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Simultaneous Recording ◽  
Periodic Paralysis ◽  
Resting Potential ◽  
Charge Movement ◽  
Vigorous Exercise ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
Gating Charge ◽  
Rapid Kinetics

Excitation-contraction coupling (ECC) is the process by which electrical excitation of muscle is converted into force generation. Depolarization of skeletal muscle resting potential contributes to failure of ECC in diseases such as periodic paralysis, intensive care unit acquired weakness and possibly fatigue of muscle during vigorous exercise. When extracellular K+ is raised to depolarize the resting potential, failure of ECC occurs suddenly, over a narrow range of resting potentials. Simultaneous imaging of Ca2+ transients and recording of action potentials (APs) demonstrated failure to generate Ca2+ transients when APs peaked at potentials more negative than –30mV. An AP property that closely correlated with failure of the Ca2+ transient was the integral of AP voltage with respect to time. Simultaneous recording of Ca2+ transients and APs with electrodes separated by 1.6mm revealed AP conduction fails when APs peak below –21mV. We hypothesize propagation of APs and generation of Ca2+ transients are governed by distinct AP properties: AP conduction is governed by AP peak, whereas Ca2+ release from the sarcoplasmic reticulum is governed by AP integral. The reason distinct AP properties may govern distinct steps of ECC is the kinetics of the ion channels involved. Na channels, which govern propagation, have rapid kinetics and are insensitive to AP width (and thus AP integral) whereas Ca2+ release is governed by gating charge movement of Cav1.1 channels, which have slower kinetics such that Ca2+ release is sensitive to AP integral. The quantitative relationships established between resting potential, AP properties, AP conduction and Ca2+ transients provide the foundation for future studies of failure of ECC induced by depolarization of the resting potential.

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