Download - Chloride Channels - Joseph M. Breza -
![Page 1: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/1.jpg)
Chloride Channels
- Joseph M. Breza -
![Page 2: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/2.jpg)
• Membrane potential– Resting potential– Facilitate fast depolarization (OFSNs)– Hyperpolarization (GABA, Glycine)
• Spike timing (ISI, bursts)
• Regulation of cell volume
• Ubiquitously expressed throughout the body and nervous system- Olfaction, Taste, Vision, Somatosensory, Auditory, Muscle, Gut
Chloride Channel Functions
![Page 3: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/3.jpg)
• Voltage
• Volume (swelling)
• Ligand Binding
• Ion Concentration
• ATP
• Protonation
• Phosphorylation
Gating Mechanisms
![Page 4: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/4.jpg)
Unlike K Channels, Chloride channels are less understood.
CaC and CaK channels are frequently coexpressed and coactivated by Ca2+ and help to stabilize membrane potentials
Interestingly, prokaryotic ClC channels function more as H+/Cl- transporters ratherThan anion channels
ClC channels are far more complex then K channels and can not be predicted by Hydrophobic analysis
- Many possible ion pores are hidden in channels
- HEK cells and Oocytes have Cl- channels
In general, Cl- channel blockers are dirty and can block cation current as well
![Page 5: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/5.jpg)
ClC-2 – activated by hyperpolarization, acidic pH and swelling.KO results in retinal degeneration or male infertility and spontaneous seizures.
Mutations in chloride channels
ClC-1 - Myotonia Congenita (neuromuscular disorder) ~ 75% of resting conductance.- shift in voltage dependency- prolonged depolarization
CFTR- Cystic fibrosis transmembrane conductance regulator - thick mucous production- effects the lungs, digestive and immune systems
ClC-Kb - Bartter syndrome- low K+ levels- alkalosis- low blood pressure
VomitingDehydrationElectrolyte imbalance
![Page 6: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/6.jpg)
Suzuki et al 2006
Chloride channel types
Ca2+ activated Cl- channels 4 TMSs
5-6 TMSs 1 TMS
12 TMSs With nucleotideBinding domainsAnd a regulatory domain
Assumed to have 10-12 TMSs
Crystallography suggests18 a-helices
![Page 7: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/7.jpg)
Na+
K+
Cl-
Na+
Na+
Ca++
Na+
Na+ Na+
Na+
Na+
K+
K+ K+
K+
K+
K+K+K+
Cl-
Cl-Cl-
Cl-
Cl-
Cl-
Cl-
Ca++ Ca++
Ca++
Ca++
Ca++
Stabilization of Membrane Potential
![Page 8: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/8.jpg)
Na+
K+
Cl-
Na+
Na+
Ca++
Na+
Na+ Na+
Na+
Na+
K+
K+ K+
K+
K+
K+K+K+
Cl-Cl-
Cl- Cl-
Cl-
Cl-
Cl-
Ca++ Ca++
Ca++
Ca++
Ca++
Stabilization of Membrane Potential
![Page 9: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/9.jpg)
Whole Cell Patch Clamp
![Page 10: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/10.jpg)
Inhibition of Skeletal Muscle ClC-1 Chloride Channels by Low Intracellular pH and ATP
Brett Bennetts, Michael W. Parker & Brett A. Cromer J Biol Chem. 2007 [Epub ahead of print]
![Page 11: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/11.jpg)
pH 7.2
pH 7.2 + 1mM ATP
pH 6.2
pH 6.2 + 1mM ATP
Bennetts et al 2007
Effect of pH on Open Probability
![Page 12: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/12.jpg)
Bennetts et al 2007
CBS domains ClC-1Key residue of ATP common gating
![Page 13: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/13.jpg)
pH 7.2
pH 7.2 + 5mM ATP
pH 6.2
pH 6.2 + 5mM ATP
His847Ala His847ArgpH 7.9
Independent effects of pH and ATP are abolished
Role of Histidine Residues in Common Gating
Effect of ATP on common gating is abolishedpH + ATP effect is reduced
Bennetts et al 2007
![Page 14: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/14.jpg)
pH 7.2
pH 7.2 + 1mM ATP
pH 6.2
pH 6.2 + 1mM ATP
pH 7.9
Not significantlyDifferent than wild type
Role of Histidine Residues in Common Gating
His835Ala
Bennetts et al 2007
![Page 15: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/15.jpg)
1) His847 and His835 (protonatable residues) are important in the effect of intracellular acidosis on ClC-1 common gating.
2) His847 is important for independent effects of protons and ATP.- likely to be involved in the cooperative actions between intracellular acidosis and ATP.
3) His835Ala mutation separates the ATP effect, but not the synergistic effect of acidosis and ATP.
Summary
![Page 16: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/16.jpg)
Characterization of a Novel Voltage-Dependent Outwardly Rectifying Anion Current in Caenorhabditis Elegans Oocytes.
Am J Physiol Cell Physiol 292(1):C269-77, 2007
Xiaoyan Yin, Jerod Denton, Xiaohui Yan and Kevin Strange
![Page 17: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/17.jpg)
Background current unknown source
Outwardly Rectifying Chloride Channel (ICl,OR) CLH-3 KO
Whole cell patch
Yin et al 2006
![Page 18: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/18.jpg)
Yin et al 2006
Open Probability
![Page 19: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/19.jpg)
Yin et al 2006
Effect of Zinc and Low pH on Current
![Page 20: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/20.jpg)
Yin et al 2006 (modified)
Channel Selectivity80
-80
0
![Page 21: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/21.jpg)
Summary
• Outward rectification is due to voltage-dependent current activation at depolarized voltages.
• SCN- > I- > Br- > Cl- > F-
• Inhibited by Zinc and low pH (4.8)
• Rapidly inactivates at voltages more hyperpolarized than ~20 mV.
![Page 22: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/22.jpg)
Calcium-activated Chloride Conductance in Frog Olfactory Cilia
Steven J. Kleene and Robert C. Gesteland
The Journal of Neuroscience (11): 3624-3629], 1991
![Page 23: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/23.jpg)
Northern Grass Frog Rana Pipiens
OSN
Chloride Channels in Olfaction
![Page 24: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/24.jpg)
Cytoplasmic end
Extracellular end
Ciliary Patch Configuration
![Page 25: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/25.jpg)
Kleene and Gesteland, 1991
Effect of Cytoplasmic Ca2+ on Membrane Conductance
Current-voltage relationship
Ca2+ concentration
![Page 26: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/26.jpg)
Kleene and Gesteland, 1991
Effect of Cytoplasmic Ca2+ on Membrane Conductancew/o Na+ and K+
![Page 27: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/27.jpg)
Kleene and Gesteland, 1991
Percent of Cl- replaced by Gluconate
Chloride Dependence on Ca2+ Activated Ciliary Conductance
Reversal potentialShifted to negative voltages
![Page 28: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/28.jpg)
Kleene and Gesteland, 1991
Inhibition of Ca2+ Activated Cl- Current by DCDPC
DCDPC concentration
![Page 29: Chloride Channels - Joseph M. Breza -](https://reader033.vdocument.in/reader033/viewer/2022051008/5a4d1b597f8b9ab0599aa56f/html5/thumbnails/29.jpg)
• Ciliary conductance increases with an increase in cytoplasmic Ca2+
• Most of the Ca2+ activated current is carried by Cl-
• The Ca2+ activated current persists in the absence of Na+ and K+
• The Cl- channel inhibitor DCDPC reduces the Ca2+ activated current by 90%
Summary