Transcript Molecular Structure and Physiological Function of Chloride

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
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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
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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
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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
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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
Y578 Mutations Alter
Gating of CLC-1
Tyrosine (Y) nonpolar
Alanine (A) nonpolar
Phenylalanine (F) nonpolar
Histidine, positive charge
Lysine (K), positive charge
Glutamic Acid (E) negative charge
Most severe effect seen in Y578E,
but was unable to fit to curve
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 Zn
Y578A and Y578F are nonpolar
Y578E is negatively charged
Above mutations negate favorable
interactions with E232
Inhibited by Zn
Y578K 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 NAD
Closed = 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 NAD
Closed = 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 = uncharged
Aspartic 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
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
G551D-CFTR decay = 31.1 s s
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
ATP Binds
at Site 2
ATP Binds at Site 1
Dissociation of ATP
at Site 2
Dissociation of
ATP at Site 1
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