Chloride Channels Joseph M Breza Chloride Channel Functions

Chloride Channels - Joseph M. Breza -

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

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

Unlike K Channels, Chloride channels are less understood. Cl. C 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 Ca. C and Ca. K channels are frequently coexpressed and coactivated by Ca 2+ and help to stabilize membrane potentials In general, Cl- channel blockers are dirty and can block cation current as well Interestingly, prokaryotic Cl. C channels function more as H+/Cl- transporters rather Than anion channels

Mutations in chloride channels Cl. C-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 Cl. C-Kb - Bartter syndrome - low K+ levels - alkalosis - low blood pressure Vomiting Dehydration Electrolyte imbalance Cl. C-2 – activated by hyperpolarization, acidic p. H and swelling. KO results in retinal degeneration or male infertility and spontaneous seizures.

Chloride channel types 5 -6 TMSs 1 TMS Ca 2+ activated Cl- channels 4 TMSs Assumed to have 10 -12 TMSs With nucleotide Binding domains And a regulatory domain Suzuki et al 2006 Crystallography suggests 18 a-helices

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

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

Whole Cell Patch Clamp

Inhibition of Skeletal Muscle Cl. C-1 Chloride Channels by Low Intracellular p. H and ATP Brett Bennetts, Michael W. Parker & Brett A. Cromer J Biol Chem. 2007 [Epub ahead of print]

Effect of p. H on Open Probability p. H 7. 2 + 1 m. M ATP p. H 6. 2 + 1 m. M ATP Bennetts et al 2007

CBS domains Cl. C-1 Bennetts et al 2007 Key residue of ATP common gating

Role of Histidine Residues in Common Gating p. H 7. 9 p. H 7. 2 His 847 Ala His 847 Arg p. H 7. 2 + 5 m. M ATP p. H 6. 2 + 5 m. M ATP Effect of ATP on common gating is abolished p. H + ATP effect is reduced Independent effects of p. H and ATP are abolished Bennetts et al 2007

Role of Histidine Residues in Common Gating His 835 Ala p. H 7. 9 p. H 7. 2 + 1 m. M ATP p. H 6. 2 + 1 m. M ATP Not significantly Different than wild type Bennetts et al 2007

Summary 1) His 847 and His 835 (protonatable residues) are important in the effect of intracellular acidosis on Cl. C-1 common gating. 2) His 847 is important for independent effects of protons and ATP. - likely to be involved in the cooperative actions between intracellular acidosis and ATP. 3) His 835 Ala mutation separates the ATP effect, but not the synergistic effect of acidosis and ATP.

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): C 269 -77, 2007

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

Open Probability Yin et al 2006

Effect of Zinc and Low p. H on Current Yin et al 2006

Channel Selectivity 80 0 -80 Yin et al 2006 (modified)

Summary • Outward rectification is due to voltage-dependent current activation at depolarized voltages. • Rapidly inactivates at voltages more hyperpolarized than ~20 m. V. • SCN- > I- > Br- > Cl- > F- • Inhibited by Zinc and low p. H (4. 8)

Calcium-activated Chloride Conductance in Frog Olfactory Cilia Steven J. Kleene and Robert C. Gesteland The Journal of Neuroscience (11): 3624 -3629], 1991

Chloride Channels in Olfaction OSN Northern Grass Frog Rana Pipiens

Ciliary Patch Configuration Cytoplasmic end Extracellular end

Effect of Cytoplasmic Ca 2+ on Membrane Conductance Current-voltage relationship Ca 2+ concentration Kleene and Gesteland, 1991

Effect of Cytoplasmic Ca 2+ on Membrane Conductance w/o Na+ and K+ Kleene and Gesteland, 1991

Chloride Dependence on Ca 2+ Activated Ciliary Conductance Percent of Cl- replaced by Gluconate Reversal potential Shifted to negative voltages Kleene and Gesteland, 1991

Inhibition of Ca 2+ Activated Cl- Current by DCDPC concentration Kleene and Gesteland, 1991

Summary • Ciliary conductance increases with an increase in cytoplasmic Ca 2+ • Most of the Ca 2+ activated current is carried by Cl- • The Ca 2+ activated current persists in the absence of Na+ and K+ • The Cl- channel inhibitor DCDPC reduces the Ca 2+ activated current by 90%