Pore-Lining Residues Identified by Single Channel SCAM Studies in Cx46 Hemichannels

2003 ◽  
Vol 10 (4) ◽  
pp. 193-199 ◽  
Author(s):  
J. Kronengold ◽  
E. B. Trexler ◽  
F. F. Bukauskas ◽  
T. A. Bargiello ◽  
V. K. Verselis
Keyword(s):  
Single Channel ◽  
Pore Lining

scholarly journals Pore-Lining Residues Identified by Single Channel SCAM Studies in Cx46 Hemichannels

2003 ◽  
Vol 10 (4-6) ◽  
pp. 193-199 ◽  
Author(s):  
J. Kronengold ◽  
E. B. Trexler ◽  
F. F. Bukauskas ◽  
T. A. Bargiello ◽  
V. K. Verselis
Keyword(s):  
Single Channel ◽  
Pore Lining

scholarly journals Differentially altered Ca2+ regulation and Ca2+ permeability in Cx26 hemichannels formed by the A40V and G45E mutations that cause keratitis ichthyosis deafness syndrome

2010 ◽  
Vol 136 (1) ◽  
pp. 47-62 ◽  
Author(s):  
Helmuth A. Sánchez ◽  
Gülistan Meşe ◽  
Miduturu Srinivas ◽  
Thomas W. White ◽  
Vytas K. Verselis
Keyword(s):  
Single Channel ◽  
Sensorineural Deafness ◽  
Cell Integrity ◽  
Extracellular Loop ◽  
Amino Terminal ◽  
Cysteine Substitution ◽  
Common Causes ◽  
Excessive Entry ◽  
Underlying Mechanisms ◽  
Pore Lining

Mutations in GJB2, which encodes Cx26, are one of the most common causes of inherited deafness in humans. More than 100 mutations have been identified scattered throughout the Cx26 protein, most of which cause nonsyndromic sensorineural deafness. In a subset of mutations, deafness is accompanied by hyperkeratotic skin disorders, which are typically severe and sometimes fatal. Many of these syndromic deafness mutations localize to the amino-terminal and first extracellular loop (E1) domains. Here, we examined two such mutations, A40V and G45E, which are positioned near the TM1/E1 boundary and are associated with keratitis ichthyosis deafness (KID) syndrome. Both of these mutants have been reported to form hemichannels that open aberrantly, leading to “leaky” cell membranes. Here, we quantified the Ca2+ sensitivities and examined the biophysical properties of these mutants at macroscopic and single-channel levels. We find that A40V hemichannels show significantly impaired regulation by extracellular Ca2+, increasing the likelihood of aberrant hemichannel opening as previously suggested. However, G45E hemichannels show only modest impairment in regulation by Ca2+ and instead exhibit a substantial increase in permeability to Ca2+. Using cysteine substitution and examination of accessibility to thiol-modifying reagents, we demonstrate that G45, but not A40, is a pore-lining residue. Both mutants function as cell–cell channels. The data suggest that G45E and A40V are hemichannel gain-of-function mutants that produce similar phenotypes, but by different underlying mechanisms. A40V produces leaky hemichannels, whereas G45E provides a route for excessive entry of Ca2+. These aberrant properties, alone or in combination, can severely compromise cell integrity and lead to increased cell death.

scholarly journals Mechanical gating of the auditory transduction channel TMC1 involves the fourth and sixth transmembrane helices

2022 ◽  
Author(s):  
Nurunisa Akyuz ◽  
K. Domenica Karavitaki ◽  
Bifeng Pan ◽  
Panos I. Tamvakologos ◽  
Kelly P. Brock ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Hair Cells ◽  
Single Channel ◽  
Transmembrane Helices ◽  
Open Probability ◽  
Transmembrane Channel ◽  
Force Sensitivity ◽  
Electrophysiological Recordings ◽  
Auditory Transduction ◽  
Pore Lining

The transmembrane channel-like (TMC) 1 and 2 proteins play a central role in auditory transduction, forming ion channels that convert sound into electrical signals. However, the molecular mechanism of their gating remains unknown. Here, using predicted structural models as a guide, we probed the effects of twelve mutations on the mechanical gating of the transduction currents in native hair cells of Tmc1/2-null mice expressing virally introduced TMC1 variants. Whole-cell electrophysiological recordings revealed that mutations within the pore-lining transmembrane (TM) helices 4 and 6 modified gating, reducing the force sensitivity or shifting the open probability of the channels, or both. For some of the mutants, these changes were accompanied by a change in single-channel conductance. Our observations are in line with a model wherein conformational changes in the TM4 and TM6 helices are involved in the mechanical gating of the transduction channel.

scholarly journals Single-channel SCAM Identifies Pore-lining Residues in the First Extracellular Loop and First Transmembrane Domains of Cx46 Hemichannels

2003 ◽  
Vol 122 (4) ◽  
pp. 389-405 ◽  
Author(s):  
J. Kronengold ◽  
E.B. Trexler ◽  
F.F. Bukauskas ◽  
T.A. Bargiello ◽  
V.K. Verselis
Keyword(s):  
Transmembrane Domain ◽  
Single Channel ◽  
Extracellular Loop ◽  
Substituted Cysteine Accessibility ◽  
Charge Selectivity ◽  
Complementary Region ◽  
Excised Patches ◽  
Opposing Effects ◽  
Pore Lining ◽  
Positively Charged

Gap junction (GJ) channels provide an important pathway for direct intercellular transmission of signaling molecules. Previously we showed that fixed negative charges in the first extracellular loop domain (E1) strongly influence charge selectivity, conductance, and rectification of channels and hemichannels formed of Cx46. Here, using excised patches containing Cx46 hemichannels, we applied the substituted cysteine accessibility method (SCAM) at the single channel level to residues in E1 to determine if they are pore-lining. We demonstrate residues D51, G46, and E43 at the amino end of E1 are accessible to modification in open hemichannels to positively and negatively charged methanethiosulfonate (MTS) reagents added to cytoplasmic or extracellular sides. Positional effects of modification along the length of the pore and opposing effects of oppositely charged modifying reagents on hemichannel conductance and rectification are consistent with placement in the channel pore and indicate a dominant electrostatic influence of the side chains of accessible residues on ion fluxes. Hemichannels modified by MTS-EA+, MTS-ET+, or MTS-ES− were refractory to further modification and effects of substitutions with positively charged residues that electrostatically mimicked those caused by modification with the positively charged MTS reagents were similar, indicating all six subunits were likely modified. The large reductions in conductance caused by MTS-ET+ were visible as stepwise reductions in single-channel current, indicative of reactions occurring at individual subunits. Extension of single-channel SCAM using MTS-ET+ into the first transmembrane domain, TM1, revealed continued accessibility at the extracellular end at A39 and L35. The topologically complementary region in TM3 showed no evidence of reactivity. Structural models show GJ channels in the extracellular gap to have continuous inner and outer walls of protein. If representative of open channels and hemichannels, these data indicate E1 as constituting a significant portion of this inner, pore-forming wall, and TM1 contributing as pore-lining in the extracellular portion of transmembrane span.

scholarly journals Charged Pore-lining Residues are Required for Normal Channel Kinetics in the Eukaryotic Mechanosensitive Ion Channel MSL1

2020 ◽  
Author(s):  
Angela M. Schlegel ◽  
Elizabeth S. Haswell
Keyword(s):  
Ion Channels ◽  
Single Channel ◽  
Clear Correlation ◽  
Inner Mitochondrial Membrane ◽  
E Coli ◽  
State Expansion ◽  
Channel Characteristics ◽  
Mechanosensitive Ion Channel ◽  
Patch Clamp Electrophysiology ◽  
Pore Lining

ABSTRACTMechanosensitive (MS) ion channels are widespread mechanisms for cellular mechanosensation that can be directly activated by membrane tension. The well-studied MscS family of MS ion channels is found in bacteria, archaea, and plants. MscS-Like (MSL)1 is localized to the inner mitochondrial membrane of Arabidopsis thaliana, where it is required for normal mitochondrial responses to oxidative stress. Like Escherichia coli MscS, MSL1 has a pore-lining helix that is kinked. However, in MSL1 this kink is comprised of two charged pore-lining residues, R326 and D327. Using single channel patch-clamp electrophysiology in E. coli, we show that altering the size and charge of R326 and D327 leads to dramatic changes in open state dwell time. Modest changes in gating pressure and open state stability were also observed while no effects on channel rectification or conductance were detected. MSL1 channel variants had differing physiological function in E. coli hypoosmotic shock assays, without clear correlation between function and particular channel characteristics. Taken together, these results demonstrate that altering pore-lining residue charge and size disrupts normal channel state stability and gating transitions, and led us to propose the “sweet spot” model. In this model, the transition to the closed state is facilitated by attraction between R326 and D327 and repulsion between R326 residues of neighboring monomers. In the open state, expansion of the channel reduces inter-monomeric repulsion, rendering open state stability influenced mainly by attractive forces. This work provides insight into how unique charge-charge interactions can be combined with an otherwise conserved structural feature to help modulate MS channel function.

Data Acquisition and Handling Unit for a Quantitative Use of Electron Energy Loss Spectroscopy

1977 ◽  
Vol 35 ◽  
pp. 232-233 ◽  
Author(s):  
P. Trebbia ◽  
P. Ballongue ◽  
C. Colliex
Keyword(s):  
Electron Microscope ◽  
Energy Loss ◽  
Electron Energy ◽  
Electron Energy Loss Spectroscopy ◽  
Single Channel ◽  
Detection System ◽  
Surface Barrier ◽  
Solid State Detector ◽  
Electrostatic Mirror ◽  
Electron Energy Loss

An effective use of electron energy loss spectroscopy for chemical characterization of selected areas in the electron microscope can only be achieved with the development of quantitative measurements capabilities.The experimental assembly, which is sketched in Fig.l, has therefore been carried out. It comprises four main elements.The analytical transmission electron microscope is a conventional microscope fitted with a Castaing and Henry dispersive unit (magnetic prism and electrostatic mirror). Recent modifications include the improvement of the vacuum in the specimen chamber (below 10-6 torr) and the adaptation of a new electrostatic mirror.The detection system, similar to the one described by Hermann et al (1), is located in a separate chamber below the fluorescent screen which visualizes the energy loss spectrum. Variable apertures select the electrons, which have lost an energy AE within an energy window smaller than 1 eV, in front of a surface barrier solid state detector RTC BPY 52 100 S.Q. The saw tooth signal delivered by a charge sensitive preamplifier (decay time of 5.10-5 S) is amplified, shaped into a gaussian profile through an active filter and counted by a single channel analyser.

Instrumentation for Measurement of Lingual Strength

1968 ◽  
Vol 11 (1) ◽  
pp. 189-193 ◽  
Author(s):  
Lois Joan Sanders
Keyword(s):  
Single Channel ◽  
Displacement Transducer ◽  
Tongue Pressure ◽  
Maximum Pressure ◽  
Research Use ◽  
Direct Writing ◽  
Significant Difference ◽  
Pressure Unit ◽  
Force Displacement ◽  
Consistent Response

A tongue pressure unit for measurement of lingual strength and patterns of tongue pressure is described. It consists of a force displacement transducer, a single channel, direct writing recording system, and a specially designed tongue pressure disk, head stabilizer, and pressure unit holder. Calibration with known weights indicated an essentially linear and consistent response. An evaluation of subject reliability in which 17 young adults were tested on two occasions revealed no significant difference in maximum pressure exerted during the two test trials. Suggestions for clinical and research use of the instrumentation are noted.

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