1. Chloride Channels
- Joseph M. Breza -

2. Chloride Channel Functions
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

3. Gating Mechanisms Voltage Volume (swelling) Ligand Binding
Ion Concentration
ATP
Protonation
Phosphorylation

4. Unlike K Channels, Chloride channels are less understood.
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
CaC and CaK channels are frequently coexpressed and coactivated by Ca2+ and help to stabilize membrane potentials
In general, Cl- channel blockers are dirty and can block cation current as well
Interestingly, prokaryotic ClC channels function more as H+/Cl- transporters rather
Than anion channels

5. 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
Vomiting
Dehydration
Electrolyte imbalance
ClC-2 – activated by hyperpolarization, acidic pH and swelling.
KO results in retinal degeneration or male infertility and spontaneous seizures.

6. Chloride channel types
5-6 TMSs
1 TMS
Ca2+ activated
Cl- channels 4 TMSs
Assumed to have
10-12 TMSs
12 TMSs
With nucleotide
Binding domains
And a regulatory domain
Crystallography suggests
18 a-helices
Suzuki et al 2006

7. Stabilization of Membrane Potential
Na+
Na+
Na+
Na+
Na+
Na+
Ca++
Ca++
Ca++
Ca++
Ca++
Na+
Ca++
Na+
Cl-
Cl- K+
Cl-
Cl- K+
Cl-
Cl- K+
Cl- K+ K+ K+ K+
Cl- K+ K+

8. Stabilization of Membrane Potential
Na+
Na+
Na+
Na+
Na+
Na+
Ca++
Ca++
Ca++
Ca++
Ca++
Na+
Ca++
Na+
Cl-
Cl- K+
Cl- K+
Cl-
Cl-
Cl- K+
Cl- K+ K+
Cl- K+ K+ K+ K+

9. Whole Cell Patch Clamp

10. Inhibition of Skeletal Muscle ClC-1 Chloride Channels by
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 [Epub ahead of print]

11. Effect of pH on Open Probability
pH mM ATP
pH 6.2
pH mM ATP
Bennetts et al 2007

12. CBS domains ClC-1
Key residue of ATP common gating
Bennetts et al 2007

13. Role of Histidine Residues in Common Gating
His847Ala
pH 7.9
His847Arg
pH 7.2
pH mM ATP
pH 6.2
pH mM ATP
Effect of ATP on common gating is abolished
pH + ATP effect is reduced
Independent effects of pH and ATP are abolished
Bennetts et al 2007

14. Role of Histidine Residues in Common Gating
pH 7.9
His835Ala
pH 7.2
pH mM ATP
pH 6.2
pH mM ATP
Not significantly
Different than wild type
Bennetts et al 2007

15. Summary
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.

16. Characterization of a Novel Voltage-Dependent Outwardly Rectifying Anion Current in Caenorhabditis Elegans Oocytes.
Xiaoyan Yin, Jerod Denton, Xiaohui Yan and Kevin Strange
Am J Physiol Cell Physiol 292(1):C269-77, 2007

17. Outwardly Rectifying Chloride Channel (ICl,OR) CLH-3 KO
Whole cell patch
Background current unknown source
Yin et al 2006

18. Open Probability
Yin et al 2006

19. Effect of Zinc and Low pH on Current
Yin et al 2006

20. Channel Selectivity 80
-80
Yin et al 2006 (modified)

21. Summary
Outward rectification is due to voltage-dependent current activation at depolarized voltages.
Rapidly inactivates at voltages more hyperpolarized than ~20 mV.
SCN- > I- > Br- > Cl- > F-
Inhibited by Zinc and low pH (4.8)

22. Calcium-activated Chloride Conductance in Frog Olfactory Cilia
Steven J. Kleene and Robert C. Gesteland
The Journal of Neuroscience (11): ], 1991

23. Chloride Channels in Olfaction
OSN
Northern Grass Frog Rana Pipiens

24. Ciliary Patch Configuration
Cytoplasmic end
Extracellular end

25. Effect of Cytoplasmic Ca2+ on Membrane Conductance
Current-voltage relationship
Ca2+ concentration
Kleene and Gesteland, 1991

26. Effect of Cytoplasmic Ca2+ on Membrane Conductance w/o Na+ and K+
Kleene and Gesteland, 1991

27. Chloride Dependence on Ca2+ Activated Ciliary Conductance
Percent of
Cl- replaced
by Gluconate
Reversal potential
Shifted to negative voltages
Kleene and Gesteland, 1991

28. Inhibition of Ca2+ Activated Cl- Current by DCDPC
concentration
Kleene and Gesteland, 1991

29. Summary
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%