scholarly journals Properties of channels reconstituted from the major intrinsic protein of lens fiber membranes.

1990 ◽  
Vol 96 (3) ◽  
pp. 631-664 ◽  
Author(s):  
G R Ehring ◽  
G Zampighi ◽  
J Horwitz ◽  
D Bok ◽  
J E Hall
Keyword(s):  
Lipid Bilayers ◽  
High Performance ◽  
Single Channel ◽  
Freeze Fracture ◽  
Boltzmann Distribution ◽  
Major Intrinsic Protein ◽  
Time Constants ◽  
Intrinsic Protein ◽  
Lens Fibers ◽  
Conductance States

Detergent-solubilized plasma membrane protein of either adult bovine or calf lens and high-performance liquid chromatography-purified major intrinsic protein (MIP) of the lens were reconstituted into unilamellar vesicles and planar lipid bilayers. Freeze-fracture studies showed that the density of intramembrane particles in the vesicles was proportional to the protein/lipid ratio. At high ratios, these particles crystallized into tetragonal arrays as does MIP in lens fibers. Channels induced by either purified MIP or detergent-solubilized protein had essentially identical properties. The conductance of multichannel membranes was maximal near 0 mV and decreased to 0.49 +/- 0.08 of the maximum value at voltages greater than 80 mV. The dependence of the conductance on voltage was well fit by a two-state Boltzmann distribution. Voltage steps greater than 30 mV elicited an ohmic current step followed by a slow (seconds) biexponential decrease. The amplitudes and time constants depended on the magnitude but not the sign of the voltage. Steps from 100 mV to voltages less than 30 mV caused the channels to open exponentially with a millisecond time constant. Analysis of latency to first closure after a voltage step gave nearly the same time constants as multichannel kinetics. Single-channel conductance is proportional to salt concentration from 0.1 to 1.0 M in KCl. In 0.1M KCl, the channel had two preferred conductance states with amplitudes of 380 and 160 pS, as well as three additional substates. Multi- and single-channel data suggest that the channel has two kinetically important open states. The channel is slightly anion selective. The properties of the channel do not vary appreciably from pH 7.4 to 5.8 or from pCa 7 to 2. We propose that a channel with these properties could contribute to maintenance of lens transparency and fluid balance.

Calcium-dependent halothane activation of sarcoplasmic reticulum calcium channels from frog skeletal muscle

1994 ◽  
Vol 266 (2) ◽  
pp. C391-C396 ◽  
Author(s):  
R. Bull ◽  
J. J. Marengo
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Calcium Channels ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Free Calcium ◽  
Frog Skeletal Muscle ◽  
Time Constants ◽  
Calcium Dependent ◽  
Open Time

The effect of halothane on calcium channels present in sarcoplasmic reticulum membranes isolated from frog skeletal muscle was studied at the single channel level after fusing the isolated vesicles into planar lipid bilayers. Addition of 91 microM halothane to the cytosolic compartment containing 1 microM free calcium activated the channel by increasing fractional open time from 0.11 to 0.59, without changing the channel conductance. The activation of the channels by halothane was calcium dependent. At resting calcium concentrations in the cytosolic compartment, halothane failed to activate the channel, whereas maximal activation was found at 10 microM calcium. The free energy of halothane binding to the channel decreased from -5.8 kcal/mol at 1 microM calcium to -6.6 kcal/mol at 10 microM calcium. Halothane increased the open time constants and decreased the closed time constants, indicating that it binds to both the open and the closed configurations of the channel.

scholarly journals Single-Channel Kinetics, Inactivation, and Spatial Distribution of Inositol Trisphosphate (IP3) Receptors in Xenopus Oocyte Nucleus

1997 ◽  
Vol 109 (5) ◽  
pp. 571-587 ◽  
Author(s):  
Don-On Daniel Mak ◽  
J. Kevin Foskett
Keyword(s):  
Single Channel ◽  
Inositol Trisphosphate ◽  
Boltzmann Distribution ◽  
Oocyte Nucleus ◽  
Distribution Analysis ◽  
Ip3 Receptors ◽  
Open Probability ◽  
Reversible Inactivation ◽  
Patch Clamp Electrophysiology ◽  
Conductance States

Single-channel properties of the Xenopus inositol trisphosphate receptor (IP3R) ion channel were examined by patch clamp electrophysiology of the outer nuclear membrane of isolated oocyte nuclei. With 140 mM K+ as the charge carrier (cytoplasmic [IP3] = 10 μM, free [Ca2+] = 200 nM), the IP3R exhibited four and possibly five conductance states. The conductance of the most-frequently observed state M was 113 pS around 0 mV and ∼300 pS at 60 mV. The channel was frequently observed with high open probability (mean Po = 0.4 at 20 mV). Dwell time distribution analysis revealed at least two kinetic states of M with time constants τ < 5 ms and ∼20 ms; and at least three closed states with τ ∼1 ms, ∼10 ms, and >1 s. Higher cytoplasmic potential increased the relative frequency and τ of the longest closed state. A novel “flicker” kinetic mode was observed, in which the channel alternated rapidly between two new conductance states: F1 and F2. The relative occupation probability of the flicker states exhibited voltage dependence described by a Boltzmann distribution corresponding to 1.33 electron charges moving across the entire electric field during F1 to F2 transitions. Channel run-down or inactivation (τ ∼ 30 s) was consistently observed in the continuous presence of IP3 and the absence of change in [Ca2+]. Some (∼10%) channel disappearances could be reversed by an increase in voltage before irreversible inactivation. A model for voltage-dependent channel gating is proposed in which one mechanism controls channel opening in both the normal and flicker modes, whereas a separate independent mechanism generates flicker activity and voltage- reversible inactivation. Mapping of functional channels indicates that the IP3R tends to aggregate into microscopic (<1 μm) as well as macroscopic (∼10 μm) clusters. Ca2+-independent inactivation of IP3R and channel clustering may contribute to complex [Ca2+] signals in cells.

scholarly journals Electron microscopic observations of reconstituted proteoliposomes with the purified major intrinsic membrane protein of eye lens fibers.

1987 ◽  
Vol 105 (4) ◽  
pp. 1679-1689 ◽  
Author(s):  
I Dunia ◽  
S Manenti ◽  
A Rousselet ◽  
E L Benedetti
Keyword(s):  
Plasma Membrane ◽  
Natural Fiber ◽  
Freeze Fracture ◽  
Immunogold Labeling ◽  
Membrane Domains ◽  
Major Intrinsic Protein ◽  
Electron Microscopic ◽  
Lens Fibers ◽  
Multilamellar Liposomes ◽  
Close Contacts

The purified major intrinsic protein of the lens fiber plasma membrane (MP26) reconstituted into liposomes favored membrane-to-membrane close contacts as visualized by freeze fracture and immunoelectron microscopy. Reconstituted apposed unilamellar vesicles formed pentalaminar profiles, and multilamellar liposomes showed regions of stacked bilayers. Immunogold labeling, using antibody directed against MP26, demonstrated that this polypeptide is present in regions of membrane-to-membrane close interaction. Fracture faces displayed both randomly distributed clusters of 8-nm polygonal intramembrane particles and membrane domains where a bidimensional lattice of repeating subunits was present. The structural pleomorphism which characterized the MP26-reconstituted proteoliposomes seems quite comparable to that visualized in natural fiber plasma membrane domains.

scholarly journals Batrachotoxin-modified sodium channels in planar lipid bilayers. Ion permeation and block.

1987 ◽  
Vol 89 (6) ◽  
pp. 841-872 ◽  
Author(s):  
W N Green ◽  
L B Weiss ◽  
O S Andersen
Keyword(s):  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Divalent Cations ◽  
Cation Binding ◽  
Planar Lipid Bilayers ◽  
Surface Charges ◽  
High Concentrations ◽  
Conductance States ◽  
Single Channel Currents

Batrachotoxin-modified, voltage-dependent sodium channels from canine forebrain were incorporated into planar lipid bilayers. Single-channel conductances were studied for [Na+] ranging between 0.02 and 3.5 M. Typically, the single-channel currents exhibited a simple two-state behavior, with transitions between closed and fully open states. Two other conductance states were observed: a subconductance state, usually seen at [NaCl] greater than or equal to 0.5 M, and a flickery state, usually seen at [NaCl] less than or equal to 0.5 M. The flickery state became more frequent as [NaCl] was decreased below 0.5 M. The K+/Na+ permeability ratio was approximately 0.16 in 0.5 and 2.5 M salt, independent of the Na+ mole fraction, which indicates that there are no interactions among permeant ions in the channels. Impermeant and permeant blocking ions (tetraethylammonium, Ca++, Zn++, and K+) have different effects when added to the extracellular and intracellular solutions, which indicates that the channel is asymmetrical and has at least two cation-binding sites. The conductance vs. [Na+] relation saturated at high concentrations, but could not be described by a Langmuir isotherm, as the conductance at low [NaCl] is higher than predicted from the data at [NaCl] greater than or equal to 1.0 M. At low [NaCl] (less than or equal to 0.1 M), increasing the ionic strength by additions of impermeant monovalent and divalent cations reduced the conductance, as if the magnitude of negative electrostatic potentials at the channel entrances were reduced. The conductances were comparable for channels in bilayers that carry a net negative charge and bilayers that carry no net charge. Together, these results lead to the conclusion that negative charges on the channel protein near the channel entrances increase the conductance, while lipid surface charges are less important.

scholarly journals Developmental Regulation of the Direct Interaction between the Intracellular Loop of Connexin 45.6 and the C Terminus of Major Intrinsic Protein (Aquaporin-0)

2005 ◽  
Vol 280 (23) ◽  
pp. 22081-22090 ◽  
Author(s):  
Xun Sean Yu ◽  
Xinye Yin ◽  
Eileen M. Lafer ◽  
Jean X. Jiang
Keyword(s):  
Gap Junction ◽  
Intercellular Communication ◽  
Direct Interaction ◽  
Lens Development ◽  
Major Intrinsic Protein ◽  
Intracellular Loop ◽  
Intrinsic Protein ◽  
Lens Fibers ◽  
Loop Region ◽  
C Terminus

The eye lens is dependent upon a network of gap junction-mediated intercellular communication to facilitate its homeostasis and development. Three gap junction-forming proteins are expressed in the lens of which two are in lens fibers, namely connexin (Cx) 45.6 and 56. Major intrinsic protein (MIP), also known as aquaporin-0 (AQP0), is the most abundant membrane protein in lens fibers. However, its role in the lens is not clear. Our previous studies show that MIP(AQP0) associates with gap junction plaques formed by Cx45.6 and Cx56 during the early stages of embryonic chick lens development but not in late embryonic and adult lenses. We report here that MIP(AQP0) directly interacts with Cx45.6 but not with Cx56. We further identified the intracellular loop of Cx45.6 as the interacting domain for the MIP(AQP0) C terminus. Surface plasmon resonance experiments indicated that the C-terminal domain of MIP(AQP0) interacts with two binding sites within the intracellular loop region of Cx45.6 with a KD(app) of 7.5 and 10.3 μm, respectively. The KD(app) for the full-length loop region is 7.7 μm. The cleavage at the intracellular loop of Cx45.6 was observed during lens development, and the C terminus of MIP(AQP0) did not interact with the loop-cleaved form of Cx45.6. Thus, the dissociation between these two proteins that occurs in the mature fibers of late lens development is likely caused by this cleavage. Finally this interaction had no impact on Cx45.6-mediated intercellular communication, suggesting that the Cx45.6-MIP(AQP0) interaction plays a novel unidentified role in lens fibers.

scholarly journals The OpenPicoAmp-100k : an open-source high performance amplifier for single channel recording in planar lipid bilayers

2019 ◽  
Author(s):  
V Shlyonsky ◽  
D Gall
Keyword(s):  
Ion Channel ◽  
Open Source ◽  
Lipid Bilayers ◽  
High Speed ◽  
High Performance ◽  
Printed Circuit Board ◽  
Single Channel ◽  
Circuit Board ◽  
Channel Current ◽  
Current Events

AbstractWe propose an upgraded version of our previously designed open-source lipid bilayer amplifier. This improved amplifier is now suitable both for the use in introductory courses in biophysics and neurosciences at the undergraduate level and for scientific research. Similar to its predecessor, the OpenPicoAmp-100k is designed using the common lithographic printed circuit board fabrication process and off-the-shelf electronic components. It consists of the high-speed headstage, followed by voltage-gain amplifier with built-in 6-order Bessel filter. The amplifier has a bandwidth of 100 kHz in the presence of 100 pF input membrane capacitance and is capable of measuring ion channel current with amplitudes from sub-pA and up to ±4 nA. At the full bandwidth and with a 1 GΩ transimpedance gain, the amplifier shows 12 pArms noise with an open input and 112 pArms noise in the presence of 100 pF input capacitance, while at the 5 kHz bandwidth (typical in single-channel experiments) noise amounts to 0.45 pArms and 2.11 pArms, respectively. Using an optocoupler circuit producing TTL-controlled current impulses and using 50% threshold analysis we show that at full bandwidth the amplifier has deadtimes of 3.5 µs and 5 µs at signal-to-noise ratios(SNR) of 9 and 1.7, respectively. Near 100% of true current impulses longer than 5 µs and 6.6 µs are detected at these two respective SNRs, while false event detection rate remains acceptably low. The wide bandwidth of the amplifier was confirmed in bilayer experiments with alamethicin, for which open ion channel current events shorter that 10 µs could be resolved.

Effect of Integral Proteins on Frozen Membrane Fracture

1980 ◽  
Vol 38 ◽  
pp. 756-759 ◽  
Author(s):  
S. Kirchanski ◽  
D. Branton
Keyword(s):  
Lipid Bilayers ◽  
Freeze Fracture ◽  
Covalent Bond ◽  
Erythrocyte Membranes ◽  
Glycophorin A ◽  
Experimental Protocol ◽  
Transmembrane Glycoprotein ◽  
Bond Breakage ◽  
Human Erythrocyte Membranes ◽  
Integral Proteins

We have investigated the effect of integral membrane proteins upon the fracturing of frozen lipid bilayers. This investigation has been part of an effort to develop freeze fracture labeling techniques and to assess the possible breakage of covalent protein bonds during the freeze fracture process. We have developed an experimental protocol utilizing lectin affinity columns which should detect small amounts of covalent bond breakage during the fracture of liposomes containing purified (1) glycophorin (a transmembrane glycoprotein of human erythrocyte membranes). To fracture liposomes in bulk, frozen liposomes are ground repeatedly under liquid nitrogen. Failure to detect any significant covalent bond breakage (contrary to (2)) led us to question the effectiveness of our grinding procedure in fracturing and splitting lipid bilayers.

Genome-wide Characterization of Major Intrinsic Protein (MIP) Gene Family in Brachypodium distachyon

2018 ◽  
Vol 13 (5) ◽  
pp. 536-552 ◽  
Author(s):  
Ankush Ashok Saddhe ◽  
Shweta ◽  
Kareem A. Mosa ◽  
Kundan Kumar ◽  
Manoj Prasad ◽  
...  
Keyword(s):  
Gene Family ◽  
Brachypodium Distachyon ◽  
Major Intrinsic Protein ◽  
Intrinsic Protein ◽  
Genome Wide
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