Membrane Biophysics 10/2014

Anion Channels

• Selectivity gradient• Plasma membrane; intracellular organelle

membranes• Set Resting Potential• Provide transport, excitability and inhibition• Activated by Hyperpolarization• Cell Swelling• pH Levels

Double-Barreled Structure of Cl Channel (CLC Family)

Double-Barreled Structure of Cl Channel

3-D crystal structure CLC

CLC Family Members• CLC-0; 1st to be studied• CLC-1; Skeletal muscle• CLC-2; Broadly expressed• CLC-K; Kidney epithelia and inner ear cells• CLC-3; Intracellular, synaptic vesicles and

organelles• CLC-4; Vesicular channel• CLC-5; Endosomal channel• CLC-6; Intracellular channel• CLC-7; Lysosomal channel

CLC-1

• Activity dependent• 70-80% RM• Skeletal muscle

CLC-2

• Important for cell-to-cell communication and survival in early development

• Activated by: – Hyperpolarization– Cell swelling– Acidic pH

CLC-K

• Homology of CLC-K1 and Ka and CLC-K2 and Kb ~90%

• Require barrtin

CLC-3

• Intracellular; endosomes and synaptic vesicles• Modulates Ca2+ activated Cl- currents

CLC-4; CLC-5

• Intracellular membrane• Relatively mysterious • Extreme outward rectification• Inhibition by extracellular acidic pH

Cystic Fibrosis Transmembrane Conductance Regulator

• cAMP activated• Expressed in apical membrane of many cell

types• Several phosphorylated sites required to open

channel• Regulates other ion channels

Swelling Activated Chloride Channels

• ICl,swell

• Moderate outward rectification• Likely 2nd messenger, not mechanically

activated (not time-dependent)

Ca2+-Activated Cl- Channels

• Modulate excitability with afterpotentials• Regulate tonus of smooth muscles• Signal transduction• Transepithelial transport• Range from 1-70 pS single-channel

conductances

Intracellular Chloride Channels

• Overexpression brings to PM • Little is known about the native tissue• Near dense-core vesicles

Ligand Gated Chloride Channels

• Excitatory neurotransmitter binding in early development

• Fast-inhibitory neurotransmitter binding– GABAA&C (brain), glycine (spinal cord)

GABAA

• 19 mammalian members have been isolated• Pentameres• Obscure function in nonneuronal tissue• 3 open states

GABAc

• Higher sensitivity to GABA• Smaller currents• Do not desensitize

Glycine

CLC-1 Channels

• CLC-1 contributes 70-80% of the resting membrane conductance of skeletal muscles

• CLC-1 mutations altering common gating cause myotonia congenita – genetic neuromuscular channelopathy in which an

initiated muscle contraction fails to terminate

• Bennetts and Parker used a Model of Cl-/H+ transport in a prokaryotic CLC CL-/H+ antiporter to model CLC-1

CLC Channels

• Homodimers• Each subunit has its own

separate, identical ion conducting pore

• 2 Gates regulate channel acitivty– Protopore Gate, which

regulates each individual pore

– Common gate, which regulates both pore simultaneously

Duran et al. 2010

Tyrosine (Y) nonpolar

Alanine (A) nonpolarPhenylalanine (F) nonpolarHistidine, positive chargeLysine (K), positive charge

Glutamic Acid (E) negative charge

Most severe effect seen in Y578E, but was unable to fit to curve

Y578 Mutations Alter Gating of CLC-1

Zn Interacts with the Extracellular Surface of CLC-1 to Inhibit Channel Activity

Y578 Mutations Alter CLC inhibition by Zn

E232, Glutamic Acid = negative charge

Insensitive to ZnY578A and Y578F are nonpolarY578E is negatively charged

Above mutations negate favorable interactions with E232

Inhibited by ZnY578K and Y578H have positive charged

Above mutations promote favorable interactions with E232

Salt Bridge Formation Between R300, R304 and D265

How do Y578 Mutants effect NAD + Inhibition of CLC-1 Channels?

NAD+ Metabolite Inhibits CLC1-1 Via Y578

• Y578 mutants were unaffected by 3mM NAD+ whereas WT was inhibited

Open Symbols = no NADClosed = 3mM NAD

• Mutations to Y578 alter CBS interactions with CLC-1

Charge Swap mutations made to residues that form salt bridges

K195D -- Lysine (+) to Aspartic Acid (-)D579K --- Aspartic Acid (-) to Lysine (+)

Small effects seen on protopore gating

Open Symbols = no NADClosed = 3mM NAD

K195D -- Lysine (+) to Aspartic Acid (-)D579K --- Aspartic Acid (-) to Lysine (+)

Background

Evidence for Ano2 KO

No Transient Cl- Currents Remain in Ano2-/-

No Transient Cl- Currents Remain in Ano2-/-

Electro-Olfactogram

Functional EOG Recordings in Fluid Phase

Functional EOG Recording in Air Phase

• No significant difference between genotypes

Ano2-/- Mice Do Not Have Olfactory Deficits

Conclusions

• Ca2+-activated Cl- currents are absent from MOE in Ano2-/- mice

• Peak amplitude of olfactory epithelia responses decreased when stimulated with liquid in KO mice

• No detectable difference in air phase EOG amplitude or in animal behavior tests

• Ca2+-activated Cl- channels not essential for olfaction

• A mutation of CFTR, a chloride channel crucial to maintaining salt and water homestasis in epethial tissues, is the cause of cystic fibrosis

• CFTR is a ATP binding protien• ATP provides the energy required to open the

pore of the channel • PKA phosphorylation regulates activity of

channel

ATP Stimulates WT-CFTR

Mutation G551D alters ATP’s effect on CFTR

G551 is conserved in ABC binding proteins

Glycine = unchargedAspartic Acid = (-) charged

ATP = (-) charged

G551D mutant has a low open probability, therefore VX-770 was used to increase the open probability

Following ATP washout, a biphasic response is seen ( a rapid current increase then slow decay)

WT decay: Fast Phase = < 1s, Slow Phase = 29.6 s G551D-CFTR decay = 31.1 s s

Two phases of current decay in WT attributed to to disassociation from 2 ATP binding site. The Fast phase is attributed to site 2, which has a lower affinity for ATP. The slow phase is attributed to site one, which has a higher affinity for ATP

G551 is the second ATP binding site. Here, the decay is similar to the slow phase of the WT, indicating ATP dissociation from site 1

G551D Mutant Causes ATP Binding Site 2 to Inhibit Current

Dissociation of ATP at Site 2

Dissociation of ATP at Site 1

ATP Binds at Site 1

ATP Binds at Site 2

Reduction of [ATP] Increases Current in G551D Mutant

ATP has a higher affinity for Site 1

Without VX-770, I is very low….effect of change in [ATP] still apparent

Y1219 in Site 2 Plays a Role in ATP Binding

• To Test if G551D site 2 mutant was indeed inhibitory…

• Mutation to other nonpolar/uncharged AA– Y1219F, Y1219I, Y1219G– Known to alter ATP affinity to Site 2

Reducing ATP affinity at Site 2 Alters Current Decay

Like Charge Mutations to G551 have similar effects