Chloride Channels
- Joseph M. Breza -
• 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
• Voltage
• Volume (swelling)
• Ligand Binding
• Ion Concentration
• ATP
• Protonation
• Phosphorylation
Gating Mechanisms
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
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
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
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
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
Whole Cell Patch Clamp
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]
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
Bennetts et al 2007
CBS domains ClC-1Key residue of ATP common gating
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
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
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
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
Background current unknown source
Outwardly Rectifying Chloride Channel (ICl,OR) CLH-3 KO
Whole cell patch
Yin et al 2006
Yin et al 2006
Open Probability
Yin et al 2006
Effect of Zinc and Low pH on Current
Yin et al 2006 (modified)
Channel Selectivity80
-80
0
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.
Calcium-activated Chloride Conductance in Frog Olfactory Cilia
Steven J. Kleene and Robert C. Gesteland
The Journal of Neuroscience (11): 3624-3629], 1991
Northern Grass Frog Rana Pipiens
OSN
Chloride Channels in Olfaction
Cytoplasmic end
Extracellular end
Ciliary Patch Configuration
Kleene and Gesteland, 1991
Effect of Cytoplasmic Ca2+ on Membrane Conductance
Current-voltage relationship
Ca2+ concentration
Kleene and Gesteland, 1991
Effect of Cytoplasmic Ca2+ on Membrane Conductancew/o Na+ and K+
Kleene and Gesteland, 1991
Percent of Cl- replaced by Gluconate
Chloride Dependence on Ca2+ Activated Ciliary Conductance
Reversal potentialShifted to negative voltages
Kleene and Gesteland, 1991
Inhibition of Ca2+ Activated Cl- Current by DCDPC
DCDPC concentration
• 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