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