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. 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

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.

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

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+

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+

Whole Cell Patch Clamp

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. 2007 [Epub ahead of print]

Effect of pH on Open Probability pH 7.2 + 1mM ATP pH 6.2 pH 6.2 + 1mM ATP Bennetts et al 2007

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

Role of Histidine Residues in Common Gating His847Ala pH 7.9 His847Arg pH 7.2 pH 7.2 + 5mM ATP pH 6.2 pH 6.2 + 5mM 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

Role of Histidine Residues in Common Gating pH 7.9 His835Ala pH 7.2 pH 7.2 + 1mM ATP pH 6.2 pH 6.2 + 1mM ATP Not significantly Different than wild type Bennetts et al 2007

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.

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

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 pH on Current Yin et al 2006

Channel Selectivity 80 -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 mV. SCN- > I- > Br- > Cl- > F- Inhibited by Zinc and low pH (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 Ca2+ on Membrane Conductance Current-voltage relationship Ca2+ concentration Kleene and Gesteland, 1991

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

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

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

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%