Download - Membrane Channels
Membrane Channels
The Cell Membrane is Selective• Criteria for passage through the phospholipid bilayer:
1. Hydrophobic
2. Net zero charge
3. Nonpolar
4. Size is also a consideration• Chemicals that will NOT pass through the
phospholipid bilayer:
1. Hydrophillic
2. Charged, ionic
3. Polar
4. Size is also a consideration
• But, using the preceding criteria, many substances vital to cellular function (e.g., ions) and survival (e.g., glucose) will not gain entry into the cell!
• So, nature has come up with channels, which selectively allow certain substances to gain entry into the cell, even though they do not meet the criteria on the preceding slide.
Outside
Inside
PlasmaMembrane
Transported Enzyme Activity
Cell SurfaceIdentity Marker
Cell Surface Receptor
Cell Adhesion Attachment to the Cytoskleton
The Many Functions of Membrane Proteins
Channels are Vital
Without channels it is energetically unfavorable to move ions across a membrane –
1. the phospholipid bilayer is ~6-8 nm thick.
2. the hydrophilic head of the phospholipid molecule projects toward the cytoplasm or the extracellular fluid.
3. the hydrophobic tails of the phospholipid molecules project toward each other.
Transmembrane Transport
a) communication among neurons• neural systems: * action potential * synaptic signaling b) receptor – brain communication• heart muscle • signaling and regulatory processes
Cystic fibrosisEpilepsyDiabetesMigrainesNeurotoxins
Channel malfunction
For the Cation to Move Through the Phospholipid Bilayer…,
1) It must lose its waters of hydration so that it is not so huge and charged; requires energy to break attractive forces between the ion and the waters.
2) Energy is also required to move a charged highly hydrophilic particle into the highly hydrophobic area of the lipid bilayer that contains the “tails” of the phospholipid molecules.
3) Based on thermodynamic calculations, so much energy would be required for this process that it would never occur.
The rate of the reaction is determined by the energy of activation, the energy input required to produce the transition state.
A + B AB C + D
Reactants transition products
(substrates) state
The uncatalyzedreaction requires a higheractivation energy than the catalyzed one does. So, thelatter runs more quickly.
There is no difference in freeenergy (ΔG) betweenuncatalyzed and catalyzedreactions. The ΔG is thethermodynamic driving forcefor the reaction and determinesthe direction of the reaction.
Transition State
Channels are thermodynamically similar to Enzymes in that the former lower the Activation Energy required to move ions across
the Membrane
Ion Flow Across the Membrane
• A Chemical can move across the membrane through one of two ways:
1. Movement through the phospholipid bilayer.
2. Movement through a H2O-filled protein channel.
The rate of diffusion is determined by the “energy of activation”, the energy input required to produce the “transition state”. (Remove H2Os of hydration and/or move into hydro-phobic environ-ment.)
A + B AB C + D
Reactants transition products
(substrates) state
Movement through a channel only requires shedding of waters of hydration (energy input would be infinite to move through the bilayer). So. Diffusion occurs more quickly.
Change in free energy is the thermodynamic driving force for diffusion and determines the direction of ion movement
Transition State
Channels are thermodynamically similar to Enzymes in that the former lower the Activation Energy required to move ions across
the Membrane
ΔG determines this!!
What do we know about the structure of gated ion channels?
A. Biochemical Information –1. MWs range from 25-250 kDal.
2. They are integral membrane glycoproteins.
3. They usually consist of 2 or more subunits.
4. The genes that code for the proteins have been isolated, cloned and sequenced. These sequences have been grouped into 6-7 protein families.
5. The primary (amino acid) sequences of these channels is known.
Use of the Hydrophobicity Plots
1) Propose 3-D structures of the channels
2) Propose functions for specific regions of the channel proteins
Amino Acid Sequence Enables Ion Channel Structure Determination
Many Techniques Can be Used to Test the Proposed Functions of Portions of
the Channel Proteins1) Sequence Homologies – used to determine which
portions of primary sequences of ion channels are the same/very similar
a) Same channel from several different species – what is conserved must be critical to channel function
e.g., Ach-gated channel – Ach receptor portion of the channel is highly conserved
Many Techniques Can be Used to Test the Proposed Functions of Portions of
the Channel Proteins1) Sequence Homologies – used to determine which
portions of primary sequences of ion channels are the same/very similar (continued)
b) Different channels with the same basic function from many tissues in one species – what is conserved must be critical to channel function
e.g., Voltage gated channels (K+, Na+, Ca2+) – all have a presumed membrane-spanning region with charged AAs at each third position (voltage sensor?), while ligand-gated channels lack this structure
Many Techniques Can be Used to Test the Proposed Functions of Portions of the
Channel Proteins2) Immunocytochemistry – a) raise antibodies to a portion of the molecule thought to be
on the intracellular or extracellular surface of the membraneb) Incubate neurons with the labeled antibodiesc) Do antibodies bind to intact neurons? (Note: antibodies are
too large to fit into a channel)Yes – sequence is on extracellular surfaceNo – sequence may be in pore or on intracellular surfaced) Next step: lyse cells and repeat exp. to see if there is
binding, i.e. is it an intracellular sequence?
Many Techniques Can be Used to Test the Proposed Functions of Portions of the
Channel Proteins3) Site-directed mutagenesis – use molecular biology
techniques to modify specific regions of a channel with a predicted function – does modification alter channel function in a predicted fashion?
4) Chimaeric Channel Construction – construct a mutant channel from sequences from 2 or more channel genes – which “parent” channel does the mutant resemble?
•Most channels have this basic structure: multimeric (quarternary structure), membrane-spanning, and, by definition, have a pore running longitudinally through the structure.•Vary in the number of subunits and complexity.
Remember your amino acids?
• Primary, secondary, and tertiary structures of proteins.
• In addition, recall that multimeric proteins are formed from the attraction of individual subunits, forming the quarternary structure.
• Recall the structure and ionization of the each of the amino acid side-chains (R).
-It wouldn’t hurt if you reviewed what a pI is.
The amino acid side –chains (R)
•The primary amino acid sequence and higher –order structures determine the channel topology.
Interior of the channel will be lined with hydrophilic amino acids.
Exterior of the channel will be lined with hydrophobic amino acids.
Selectivity Filter
• Many channels are selective for only 1 or 2 different chemicals (ions, sugars, etc.).
• The K+ channel has such a filter, which is a narrow region towards the extracellular surface of the membrane.
• Two K+ ions can occupy the selectivity filter simultaneously, with a third in a H2O-filled cavity deeper in the pore.
Proposed Mechanisms for Channel Ion Selectivity
Ach receptor channel - 6.5 A in diameter
Voltage-gated Na+ channel - 4 A in diameter
Voltage-gated K+ channel – 3.3 A in diameter
Non-specific cation channel, i.e. little selectivity other than for cations
10-20 X more Na+ than K+
100 X more K+ than Na+
Proposed Mechanisms for Channel Ion Selectivity by Channels: Ionic size
Ach receptor channel - 6.5 A in diameter
Voltage-gated Na+ channel - 4 A in diameter
Voltage-gated K+ channel – 3.3 A in diameter
Non-specific cation channel, i.e. little selectivity other than for cations
10-20 X more Na+ than K+
100 X more K+ than Na+
Non-hydrated K+ ion = 2.7 A in diameter
Non-hydrated Na+ ion = 1.9 A in diameter
If ionic size explains channel selectivity, why is the K+ channel so selective for K+ since Na+ is smaller?
Proposed Mechanisms for Ion Selectivity by Channels: Ionic size
Ach receptor channel - 6.5 A in diameter
Voltage-gated Na+ channel - 4 A in diameter
Voltage-gated K+ channel – 3.3 A in diameter
Non-specific cation channel, i.e. little selectivity other than for cations
10-20 X more Na+ than K+
100 X more K+ than Na+
Hydrated K+ ion = 3.3 A in diameter
Hydrated Na+ ion = 3.3-4 A in diameter
Modified Model = perhaps channels select based on hydrated ionic radius?
(K+ is larger, has a lower charge density and so attracts fewer waters of hydration.)
Proposed Mechanisms for Ion Selectivity by Channels: Ionic size
The modified model explains K+ channel selectivity, i.e. the hydrated K+ just fits into the channel and the hydrated Na+ is too big to fit. However, how do we explain the +/- sodium channel selectivity?
A selectivity filter exists inside the channel
Proposed Mechanisms for Ion Selectivity by Channels: Ionic size
Sodium recognition site = selectivity filter
Na+
Na+
How might it work? Similar to enzymes, but much faster?
Evidence for a Selectivity Filter
If channels are simple resistors, than movement through an open channel should be a function of the concentration gradient for the ion across the membrane
Rate of ion movement = a x [ion]I/[ion]o
(current flow = diffusion)Linear relationship with slope = a
Evidence for a Selectivity Filter
Unitary current (pa) = recordings from single channels
External [Na+] mM
Observed data for Na+ channel
Expected data
Evidence for a Selectivity Filter
Data for voltage-gated Na+ channel do not fit the model of a channel as a simple resistor in the membrane. Instead, the current flow through the Na+ channel plateaus or “saturates” at high [Na+]. This relationship looks like what happens to an enzyme at high [substrate]. Perhaps some channels select ions based on the same biochemical mechanisms used by enzymes to select their substrates?
In the end, the final determinations of channel gating mechanisms and ion selectivities will come from X-ray crystallography of the purified channels.
The K+ channel is structure such that a very narrowtube through the inverted cone shape allows for only50 H2O molecules and only 2 K+ in succession.
Because they strongly repel each other, when one enters,one will be forced out.
Crystal Structure of the K+ Channel from above and from the side
Methods for Studying Ion Channels - 1
Biochemistry– agonist, antagonist or drug binding– isolation and purification– reconstitution
Molecular biology– sequencing, cloning, mutagenesis
Structural biology– microscopy, crystallography, NMR, ...
Methods for Studying Ion Channels - 2
Electrophysiology– tissue slice– extracellular recording– intracellular recording– whole-cell recording– single channel recording
Biochemistry– radioactive ion flux
Voltage clamp
Current clamp
Concentration jump
Electric eel
Na channel proteins
ProteinPurification
Pure Na channelproteins
Microsequence
Amino acid sequence of smallregion of Na channel protein
Designprobe
Oligonucleotide probe withsequence corresponding
to aa sequence
Hybridize to cDNAlibrary containingNa channel cDNA
s s s s s s s s s s s s s s s s
Isolate and sequenceNa channel cDNA
Deduce proteinsequence
AA sequenceof entire Na
channel proteinSynthesize cDNAlibrary containing
Na channel cDNA (s)
sss
sss
sss
Isolate mRNAs includingone encoding Na channel
Cloning via Protein Purification
• Positional cloning – Shaker flies
• Cloning by sequence homology- Use the same strategy as that used in preceding slide, except that the sequence on which the cloning is based comes not from the purified protein, but rather from the already-isolated cDNA.
Normal Shaker
Positional Cloning Enabled Determination of the Shaker K Channel
Heterologous Expression Systems
Typical Ion Channels with Known Structure:
Acetylcholine receptor M2 transmembrane segment
K+ channel (KCSA)
Types of ion channels: Simple pores (GA, GAP junctions) Substrate gated channels (Nicotinic receptor) Voltage-gated channels (K-channels) Pumps (ATP-synthase, K+,Na+-ATPase)
Ion Channels• ion channels in the PM of neurons and muscles contributes
to their excitability• when open - ions move down their concentration gradients• channels possess gates to open and close them• two types: gated and non-gated
2. Gated channels: open and close in response to a stimulusA. voltage-gated: open in response to change in voltage - participate in the AP
B. ligand-gated: open & close in response to particular chemical stimuli (hormone, neurotransmitter, ion)
C. mechanically-gated: open with mechanical stimulation
1. Leakage (non-gated) or Resting channels: are always open, contribute to the resting potential-nerve cells have more K+ than Na+ leakage channels -as a result, membrane permeability to K+ is higher-K+ leaks out of cell - inside becomes more negative-K+ is then pumped back in
Types of Biochemical Mechanisms that Open and Close Channels (Cont’d)
• Nt or hormone binding to receptor causes a 2nd messenger to activate a protein kinase that phosphorylates a channel and thus opens it.
• Changes in membrane potential.• Membrane deformation (e.g., mechanical
pressure).• Selectivity by charge (i.e., positively lined pore
allows anions through; negatively lined pore allows cations through).
Na+ Channels have Gates
At rest, one is closed (the activation gate) and the other is open (the inactivation gate).
Suprathreshold depolarization affects both of them.
The resting potential, recall, is generated mainly by open “resting”, non-gated K+ channels
AXON ECF
-the number of K+ channelsdramatically outnumbers thatof Na+-however, there are a few Na leak channels along the axonalmembrane
Channel Gating Mechanisms AChR: Proposed gating mechanism
(Unwin, 1995)
OpenClosed
Channel Families
• Voltage-gated• Extracellular ligand-gated• Intracellular ligand-gated• Inward rectifier• Intercellular• Other
Voltage-gated
• sodium: I, II, III, µ1, H1, PN3• potassium: KA, Kv (1-5), Kv(r), Kv(s),KSR, BKCa, IKCa, SKCa,
KM, KACh
• calcium: L, N, P, Q, T• chloride: ClC-0 - ClC-8
Extracellular ligand-gated
• nicotinic ACh (muscle): 2 (embryonic), 2 (adult)
• nicotinic ACh (neuronal): (2-10), (2-4)• glutamate: NMDA, kainate, AMPA• P2X (ATP)• 5-HT3
• GABAA: (1-6), (1-4), (1-4), , , (1-3)• Glycine
Intracellular ligand-gated
• leukotriene C4-gated Ca2+
• ryanodine receptor Ca2+
• IP3-gated Ca2+
• IP4-gated Ca2+
• Ca2+-gated K+
• Ca2+-gated non-selective cation
• Ca2+-gated Cl– • cAMP cation• cGMP cation• cAMP chloride• ATP Cl–
• volume-regulated Cl–
• arachidonic acid-activated K+
• Na+-gated K+
Inward rectifier
• Kir – 1.1-1.3– 2.1-2.4– 3.1-3.5– 4.1-4.2– 5.1– 6.1-6.2– 7.1
Intercellular
• Gap junction channels– Cx26– Cx32– Cx37– Cx40– Cx43– Cx45– Cx50– Cx56
Other
• Mechanosensitive• Mitochondrial• Nuclear• Aquaporins• Vesicular (synaptophysin)
G-protein linked receptors coupled to ion channels
• Acetylcholine (muscarinic)• Adenosine & adenine nucleotides• Adrenaline & noradrenaline• Angiotensin• Bombesin• Bradykinin• Calcitonin• Cannabinoid• Chemokine• Cholecystokinin & gastrin• Dopamine• Endothelin• Galinin• GABA (GABAB)• Glutamate (quisqualate)
• Histamine• 5-Hydroxytryptamine (1,2)• Leukotriene• Melatonin• Neuropeptide Y• Neurotensin• Odorant peptides• Opioid peptides• Platelet-activating factor• Prostanoid• Protease-activated• Tachykinins• Taste receptors• VIP• Vasopressin and oxytocin
Structure of the AChR at 4.6Å
Miyazawa et al, (1999) (left figure was modified)
Inside Cell
Outside Cell
Na+
Ca2+Cl-
K+
Schematic model of AChR ligand binding site
based on Arias, 1997
.
NH2
T
DE
NH2
Y
C
CY
WY
ACh orantagonist
W
empty site in ACh binding protein
Brejc, 2001
filled site in ACh binding protein
Brejc, 2001
K Channel Structure at 3Å Resolution
Doyle et al, 1998
Patch Clamp Recording Technique
A B C D E
electrode
cellchannel
cell- attached patch whole- cell outside- out patch
Sizing Up Ion Channels
.
Open
ClosedCurrent
Amplitudein Picoamps (pA)
OpenDuration
in Milliseconds (ms)
current
# e
ven
ts
log time
ClosedDuration
in Milliseconds (ms)
log time
time time
# e
ven
ts
# e
ven
ts
# e
ven
ts
# e
ven
ts
Gating and Permeation
What are the Biochemical Changes that Lead to Channel Gating (Opening or Closing)?
Gating involves some type of conformational change in the protein, but other than that there are few definitive answers to the question.
However, there are several general proposed models for gating.
Types of Biochemical Mechanisms that Open and Close Channels
• Conformational change occurs in a discrete area of the channel, leading to it opening.
• The entire channel changes conformation (e.g., electrical synapses).
• Ball-and-chain – type mechanism.• Nt or hormone binding causes the channel to
open.
What are the Biochemical Changes that Lead to Channel Gating (Opening or Closing)?
A. A conformation change occurs in a discrete portion of the channel -
closed
opened
Most popular model, but currently no evidence for it!
What are the Biochemical Changes that Lead to Channel Gating (Opening or Closing)?
B. Global change occurs in the channel -
closed
opened
Evidence that electrical synapses = gap junctions function in this manner
What are the Biochemical Changes that Lead to Channel Gating (Opening or Closing)?
C. A blocking portion of the channel changes position -
closed
opened
Evidence that voltage-gated channels function in this manner
What Types of Stimuli Can Lead to Gating?
A. Binding of Chemicals = Ligand-gated or Chemically-gated Channels
1) Extracellular surface – neurotransmitter or hormone binding causes opening of an ion channel
receptor protein
Neurotransmitter or hormoneBinds to
Opened channel
What Types of Stimuli Can Lead to Gating?
A. Binding of Chemicals = Ligand-gated or Chemically-gated Channels (continued)
2) Intracellular surface – a second messenger is activated by neurotransmitter or hormone binding to a receptor
receptor protein
Neurotransmitter or hormoneBinds to
2nd messenger
1) Binds directly to a channel and opens/closes it
2) Activates a protein kinase that phosphorylates the channel and opens/closes it
What Types of Stimuli Can Lead to Gating?
B. Change in Membrane Potential = Voltage-gated channels
C. Membrane Deformation = Mechanically-gated channels (ex. Sensory system where membrane stretch or pressure on the membrane is transmitted to the channel via the cytoskeleton?)
Pressure applied
What are the Proposed Mechanisms for Ion Selectivity by Channels?
A. Channels can select by charge -Cl-
Cl-
+
+
+
+
+
+
K+
K+
-
-
-
-
-
-
Anion selective channel lined with positively charged AAs
Cation selective channel lined with negatively charged AAs
What are the Proposed Mechanisms for Ion Selectivity by Channels?
B. Channels can select based on ionic size –
Experimental approach =
1) sequence data were used to propose 3-D structures of the channels and to estimate the pore diameters
2) ionic selectivities were determined using electrophysiological techniques
A Closer Look at Gating: Kinetics of Gating
Channel Gating: Two State Model
Closed kc
ko Open
d Closed dt
ko Closed kc Open
d Open
dtko Closed kc Open
Closed=1Open=0
i s RandNum<ko* dt?
Yes No
Closed=0Open=1
Yes No
i s RandNum<kc* dt?
RandNumgenerator
Channel Gating: 2 State Simulations
43210Time (ms)
43210Time (ms)
1.0
0.8
0.6
0.4
0.2
0.0
Op
en P
rob
43210Time (ms)
Slow kinetics: ko=0.5/ms kc=0.5/ms Fast kinetics: ko=5/ms kc=5/ms
Superposition
Gating Exercise
average closed time 1ko
steady state popen
k
oko kc
average open time 1kc
onset time 1kokc
Predictions
example ko (1/ms) kc (1/ms)
averageclosed time
(ms)
averageopen time
(ms)
steady-statepopen
onsettime (ms)
1 0.5 0.5 2.0 2.0 0.5 1.0
2 5.0 5.0
3 5.0 0.5
4 0.5 5.0
Closed kc
ko Open
Channel Gating: More 2 State Simulations
43210Time (ms)
1.0
0.8
0.6
0.4
0.2
0.0
Op
en P
rob
43210Time (ms)
High popen: ko=5/ms kc=0.5/ms
43210Time (ms)
Low popen: ko=0.5/ms kc=5/ms
Superposition
Channel Gating:3 State Model
d Closed
dt ko Closed kc Open
d Open
dtko Closed kc Open k+i Open k-i Inactive
dInactive dt
k+i Open k-i Inactive
Closed kc
ko Open k-i
k+i Inactive
More realistic model V-gated Na channelsopen channel blockers
Channel Gating: 3 State Simulation
50403020100time (ms)
Open
Closed
"Supervision"Open
Inactive
Closed
141210864time (ms)
Current
Burst
ko = 0.1/ms kc =0.5/ms k+i = 1/ms k-i = 5/ms
Closed kc
ko Open k-i
k+i Inactive
Channel Gating: 3 State Superposition
ko = 0.5/ms kc =0.005/ms k+i = 0.25/ms k-i = 0.025/ms
1.0
0.8
0.6
0.4
0.2
0.0
Op
en
Pro
b
50403020100
time (ms)
Popen
k+i = 0/ms k-i = 0/ms
Channel Gating Mechanisms (1)
A Q K K IEE K Q E L Q E Q Q H Q KQ Q Q Q K K R H QR D E G L G V L G A V A A M
The “Ball and Chain” model of channel inactivation gating
At depolarized potentials, the ball is more stable at the blocking conformation.
Channel Gating Mechanisms (2)AChR: Open (white) & Closed (blue) conformations
(Unwin, 1995)
Channel Gating Mechanisms (3)AChR: Proposed gating mechanism
(Unwin, 1995)
Vr is known as the: Nernst potentialreversal potentialzero current potentialequilibrium potential
Channel Permeation - Nernst
K+
Cl-–Cl-
K+ –
–
––
–
––
–
–––
–K+
Cl-Cl-
K+ –
–
––
–K+
Cl-Cl-
K+
Vr=RTzF
lncoci
58mVz log
coci
Consider a cell containing a high concentration of KClExternal [KCl] is lowConsider the cell membrane to be permeable to K+ only.
initial condition
K+
Cl-–Cl-
K+ –
–
––
–
––
–
–––
–K+
Cl-Cl-
K+ –
–
––
–K+
Cl-Cl-
K+
after some diffusion
K+
Cl-–Cl-
K+ –
–
––
–
––
–
–––
–K+
Cl-Cl-
K+ –
–
––
–K+
Cl-Cl-
K+
equilibrium
Channel Permeation: ExamplesVr = 58 mV
zlog
coci
VK = 58 mV
+1log 5 mM
150 mM
86 mV
Ion z co (mM) ci (mM) Vr (mV)
K +1 5 150 -86
Na 150 15
Cl 120 10
Ca 2 0.0001
[K]o =5 mM, [K]i=150 mM
Channel Permeation: GHKGoldman-Hodgkin-Katz Voltage Equation
Cell membrane potential depends on ion gradients and ionic permeabilities (Pi)
Vm=58 mV log PKKo PNaNao PClCliPKKi PNaNai PClClo
1. Neuron at rest (ci, co from previous slide)PK=100, PNa=5, PCl=10 Vm= 63 mV
2. Neuron during an action potential PK=100, PNa=500, PCl=10 Vm=___
Channel Permeation: Ohm's Law
Current (flow) is equal to voltage (driving force) times conductance (1/resistance)
I = (V-Vr) G
Current (I) is measured in amps (pA, nA, µA)Voltage (V) is measured in volts (mV)Conductance (G) is measured in Siemens (pS, nS)
Channel Permeation: Turnover
How much is a picoamp of current?
1pA1012A 10 12Coul
s 1 ion1.610 19Coul
107ions/s
Ion channels are enzymes that catalyze the flow of ions across cell membranes. The catalytic rate is on the order of 107 per second.
Channel Permeation: IV Curvesa) V-independent conductance:
I = (V-Vr) G, G = N popen
1.0
0.8
0.6
0.4
0.2
0.0
Op
en
Pro
bab
ilit
y
-100 -50 0 50 100
Voltage (mV)
-1000
-500
0
500
1000
Cu
rren
t (p
A)
-100 -50 0 50 100
Voltage (mV)
N=200, = 50 pS, Vr=0
Channel Permeation: IV Curvesb) V-dependent conductance
I = (V-Vr) G(V) G = N popen(V)1.0
0.8
0.6
0.4
0.2
0.0
Op
en
Pro
bab
ilit
y
-100 -50 0 50 100
Voltage (mV)
-1000
-500
0
500
1000
Cu
rren
t (p
A)
-100 -50 0 50 100
Voltage (mV)
N=200, = 50 pS, Vr=+50 mV