Download Regulation of ion channels

Membrane Transport
“Pores, Porters and Pumps”
CH353 March 4-6, 2008
Summary
• Thermodynamics and Kinetics of Membrane Transport
• Classification of Membrane Transport Proteins
– Channels, Porters, Primary Active Transporters
• Primary Active Transporters
– driven by hydrolysis of phosphoanhydride bonds
• Porters (secondary active transport & facilitated diffusion)
– driven by electrochemical potential
• Systems combining active transporters and porters
• Channels (for water and ions)
• Regulation of ion channels
– voltage and ligand gating
– action potential and synaptic function
Non-MFS Porters
• Na+ Ca2+ Exchanger (NCX1)
– member of Ca2+: cation antiporter (CaCA) family (2.A.19)
– antiporter of 3 Na+ for 1 Ca2+
• Na + Glucose Symporter
– member of solute: sodium symporter (SSS) family (2.A.21)
– symporters of 1-2 Na+ for 1 glucose
• HCO3– Cl– Exchangers
– members of anion exchanger (AE) family (2.A.31)
– antiporters and symporters
Na+ Ca2+ Exchanger (NCX1)
• Plasma membrane antiporter
regulating intracellular Ca2+ in
cardiac myocytes
• Electrogenic process: transports
3 Na+ in for 1 Ca2+ out
• Lower affinity for Ca2+ than Ca2+
ATPase pumps, but faster
• 2000 Ca2+ /sec vs. 30 Ca2+ /sec
• Lowering Ca2+ reduces strength
of heart muscle contraction
• Digoxin inhibits Na+K+ ATPase
increasing intracellular Na+,
decreasing efficiency of Na+ Ca2+
exchanger, increasing strength of
heart contraction
Na+ Ca2+ Exchanger regulated
by cytoplasmic Ca2+ binding
domain CBD1 on exchanger
Blaustein et al. 2007,
Proc Natl Acad Sci USA 104: 18349
Na+ Glucose Symporters
• secondary active transport of glucose up concentration gradient
coupled with Na+ down electrochemical gradient (electrogenic)
• 2 human isoforms:
– SGLT1 (SLC5A1): high affinity, low capacity
• symport of 2 Na+ in for 1 glucose in
• intestinal and renal absorption of glucose and galactose
• SLC5A1 deficiency: glucose-galactose malabsorption (GGM)
– SGLT2 (SLC5A2): low affinity, high capacity
• symport of 1 Na+ in for 1 glucose in
• renal glucose reabsorption
• SLC5A2 deficiency: renal glucosuria
Glucose Transport in Intestinal Epithelium
Molecules absorbed from
intestinal lumen pass
through epithelial cells
Tight junction
Transporters in epithelial
cells require correct
localization on apical or
basolateral membranes
• Apical: SGLT1
• Basolateral: GLUT2
and Na+K+ ATPase
Energetics of Na+ Glucose Transport
• Secondary active transport of glucose coupled with the
electrochemical gradient of Na+ is energetically favorable
Na+out + glucoseout → Na+in + glucosein
[Na+]out = 145 mM
∆G
(Na+
[Na+]in = 12 mM
∆y = - 50 mV
C2
transport) = RT ln ( ) + ZF∆y
C1
= -6.4 kJ/mol + -4.9 kJ/mol = -11.3 kJ/mol
At equilibrium: ∆G = ∆G (Na+ transport) + ∆G (glucose transport) = 0
For 1 mol Na+ transported per mol glucose:
[glucose]in
11.3 kJ/mol = RT ln (
)
[glucose]out
[glucose]in
= 80.7
[glucose]out
For 2 mol Na+ transported per mol glucose:
[glucose]in
22.6 kJ/mol = RT ln (
)
[glucose]out
[glucose]in
= 6520
[glucose]out
Anion Exchangers
HCO3– Cl– Exchangers (Antiporters)
• 3 human members
AE1 (SLC4A1) (erythrocytes)
– binds to carbonic anhydrase (transport metabolon)
– exchange of CO2 at tissues and lungs
AE2 (SLC4A2)
– intracellular pH regulation; linked to Na+ H+ exchanger
– exocrine secretion
AE3 (SLC4A3)
– isoforms in kidney and heart
Function of HCO3– Cl– Exchanger (AE1)
Regulation of Intracellular pH
• Respiring cell accumulate
excess acids, e.g. H2CO3
• 3 transport systems combine
to regulate pH
• Na+ H+ antiporter (↑ pH)
– Na+ in; H+ out
• Na+ HCO3– Cl– (↑ pH)
– Na+, HCO3– in; Cl– out
• HCO3– Cl– antiporter (↓ pH)
– HCO3– out; Cl– in
Intracellular pH activates
appropriate transport system
Ionophores
Non-ribosomally synthesized porters
• Cyclic compounds that collapse
membrane concentration gradients
• Diverse composition (peptides,
polyketides, polyethers, tetrolides)
• Valinomycin – cyclic peptide
K+ uniporter (K+ > Cs+ > Na+)
• Monesin – cyclic polyketide
Na+:H+ antiporter (Na+ > K+> Rb+)
• Nigericin – cylic polyketide
K+:H+ antiporter (K+ > Rb+ > Na+)
• Tetronasin – cylic polyketide
Ca2+:H+ or Mg2+:H+ antiporter
Valinomycin – K+ complex
( D-valine – L-lactate – L-valine – D-hydroxyisovalerate ) 3
Experimental Methods for Transporters
Models
• Intact cells
• Liposomes with purified or recombinant proteins
• Oocyte expression of injected RNA
Measurements
• Membrane potential
• Ion current (patch clamp)
Study Models for Transport Proteins
• Isolate cell membrane fraction
• Purify transporters from other
integral membrane proteins
• Mix solubilized transporter
prep with phospholipids
• Remove detergent and form
liposomes with transporters
• Use transporters in liposome
for experiments
Study Models for Transporters
• Harvest oocytes from
Xenopus laevis (oocytes
are ~1 mm dia)
• Transcribe in vitro RNA
from a cDNA construct
• Microinject RNA into
Xenopus oocytes
• Allow for expression of
RNA into protein
• Measure properties of
transporter [online]
Measurement of Transporter Activity
Membrane potential is measured
using:
• microelectrode inserted into cell,
• reference electrode in medium
• potentiometer for measuring
voltage and direction of current
• System is used for measuring
action potentials on neurons and
muscle cells
Measurement of Transporter Activity
Patch clamping technique
• micropipette filled with
electrolyte is placed over
transporter on membrane
• reference electrode in
medium or inserted in cell
• measure current required
for maintaining constant a
membrane potential
• applied for measuring
transport of ions
Measurement of Transporter Activity
• Variations on experimental
setup for patch clamping
muscle cell or neuron
Measurement of Transporter Activity
Interpreting patch clamping data
a) inside-out patches of muscle cell membrane clamped at
voltage slightly less than resting potential. Drops in current
(pA) indicate opening of single Na+ channels
b) patches of neuron membrane clamped at 3 different
voltages. Increases in current show opening of K+ channels.
Channel opening increases with membrane depolarization
Channels/Pores (α-Type Channels)
Characteristics
• Rapid transport without carriers near diffusion limit (107
to 109 /s)
• Selectivity: molecule, charge or ion specific
• Gating: open, closed, or inactivated
Aquaporins
• Water-specific membrane channels
• Members of major intrinsic protein (MIP) family (1.A.8)
• Aquaporin group permeated by water only
– AQP0, AQP1, AQP2, AQP4, AQP5, (AQP6 and AQP8)
• Aquaglyceroporin group permeated by water and small
solutes, e.g. anions, glycerol, urea
– AQP3, AQP7, AQP9 and AQP10
• Different permeabilities to water
– AQP0, AQP6, AQP9 and AQP10 have low permeability to water
• Tissue specific expression of aquaporins: types, proteins
/cell and cellular localization
Regulation of Aquaporins
In kidney
• AQP1 expressed in epithelial cells of proximal tubules;
absorbs water from glomular filtrate
• AQP2 expressed in principal cells; absorbs water from
collecting ducts
• Vasopressin, a antidiruetic hormone, stimulates urine
concentration by translocating AQP2 from intracellular
vesicles to the apical plasma membrane of principal cells
Structure of Aquaporins
• AQP1 is a tetramer of 28-kDa subunits, each with a
transmembrane channel 0.2 – 0.3 nm in diameter
• each subunit has 6 transmembrane helices and 2 short
helices (non-spanning)
• each short helix extends from opposite sides toward the
middle and has an Asn-Pro-Ala (NPA) motif
• NPA motifs and connecting loops form a selectivity filter
• the 2-nm x 0.28 nm diameter pore is selective for water and
allows sequential passage of several waters; H-bonds formed
with carbonyl oxygens; closely-spaced waters not permitted
• hydrogen ions (H3O+) cannot pass through water channel;
repelled by Arg and His residues and helical dipoles
Aquaporin Selectivity Filter
• Structure of AQP1 subunit with
amino acids lining water channel
• 4 water molecules are indicated
by green spheres
• Constriction region (blue arrow)
• Pseudo two-fold symmetry axis
between N78 and N194 (black
arrow)
• Amino acids H76, H182, R197
prevent H3O+ entry
• 4 water molecules too far apart
for “H+ hopping”
Sui et al. 2001, Nature 414: 872
Ion Channels
Regulation of ion channels
a) Ligand gating
b) Voltage gating (some are refractory after opening)
Ashcroft 2006, Nature 440: 440
K+ Channels
Non-gated K+ channel (KcsA)
(Streptomyces lividans)
• Tetramer of same subunits
each with
– 2 transmembrane helices
– a short helix
– an ion-sensing loop
(selectivity filter) (yellow)
• Loops from each subunit (4)
combine to form binding sites
for 4 unsolvated K+ (green)
K+ Channel Selectivity Filter
Extracellular Side
• Carbonyl oxygens coordinate 2
unsolvated K+ at one time; each
K+ is coordinated by 8 oxygens
• K+ can occupy
– positions 1 and 3 (blue) or
– positions 2 and 4 (green)
• Binding of Na+ (radius 0.95 Å)
is energetically less favorable
than binding of K+ (1.33 Å)
• Transport of K+ is 10,000 times
faster than that of Na+
Intracellular Side
4
3
2
1
K+ Channel Transport Mechanism
• Process starts with 2 K+ bound to
the selectivity filter (sites 2 & 4)
• K+ enters channel from cytosol
• K+ loses hydration near the
selectivity filter; is stabilized by
dipoles of short α helices
• K+ enters selectivity filter (site 1)
displacing the K+ at site 2 to site
3 and the K+ at site 4 to outside;
extracellular K+ is hydrated
• K+ ions at sites 1 & 3 shift to
sites 2 & 4 for next K+ to enter
4
3
2
1
Electrochemical Membrane Potential
• Na+K+ ATPase establishes
chemical gradients across the
plasma membrane with high
[K+] and low [Na+] inside
• Secondary active transport
using the Na+ gradient creates
the gradients of other ions
• Ca2+ also has active transport
• Electrogenic transport of 3 Na+
out for 2 K+ in causes some of
the membrane potential
• Most of the membrane potential
results from transport of K+ out
of the cell through K+ channels
Resting Membrane Potential
• Membrane potential is caused by the
predominant flow of K+ from the cell
• Most animal cells maintain their
membrane potential at -50 to -70 mV
relative to outside the cell
• Excitable cells (neurons and muscle
cells) transiently alter their membrane
potential, making it more positive
• Opening Na+ channels makes the
membrane potential more positive
(depolarizing the membrane)
• Opening K+ or Cl– channels makes the
membrane potential more negative
(hyperpolarizing the membrane)
Action Potentials
• Membrane potential of excitable
cells becomes transiently more
positive (depolarizes) in response
to a stimulus – action potential
• Action potential coincides with a
brief increases in permeabilities of
membrane to Na+ and K+
• Flow of Na+ out of cell depolarizes
membrane; subsequent flow of K+
out of cell repolarizes and slightly
hyperpolarizes membrane
• Action potentials are caused by
sequential opening and closing of
voltage-gated Na+ and K+ channels
Transmission of Action Potential
• Local depolarization of membrane
spreads to adjacent region (purple)
opening voltage-gated Na+ channels
• This causes further depolarization of
membrane and opening of additional
voltage-gated Na+ channels
• Na+ channels in the region of the
membrane (green) where the action
potential originated remain closed
• These channels are refractory to
opening by depolarization for
several milliseconds
• This results in the unidirectional
transmission of the action potential
Model for Voltage-Gated Na+ Channel
Experimental Support:
• voltage-gated Na+ channel is bell-shaped by electron microscopy
• opening of channel coincides with measured displacement of charged amino
acids (gating charge) – movement of positively charged S4 helices
• mutation of inactivating sequence or its binding site abolishes refractory period
S4 Family of Pore Loop Channels
• Voltage-gated ion channels (S4 family) are related to the non-gated K+
channel (homologous S5 – pore loop – S6)
• Voltage-gated K+ channels are tetramers of same 4 subunits
• Voltage-gated Na+ and Ca2+ channels have 4 domains on one subunit
Topology of Voltage-Gated Na+ Channel
• Large subunit (~260 kDa) has 4 homologous domains each with
– 6 transmembrane helices (predicted by hydrophobicity)
– a selectivity filter (pore loop), a voltage sensor (S4) and an
activation helix (S6)
• Inactivation sequence is between domains III and IV
Voltage Gating of Na+ Channels
• Nice Model, But … No 3D Structural Support
Models for Movement of Gating Charges
• Gating charges (red +) move
from inside (bottom) to outside
(top) with depolarization of
membrane
Conventional Model
• Gating charges pass through
the protein by translation or
rotation of S4 helix within a
gating pore of the protein
New Model
• Gating charges pass through
the membrane by movement of
voltage-sensor paddles on the
outside of the protein
Closed
Open
Jiang et al. 2003, Nature 423: 33
Structure of Voltage-Gated K+ Channel
• 3D structure of voltage-gated K+
channel shows voltage sensor
on a paddle – a helix-loop-helix
formed by S3b and S4
• gating charges are shown as
blue side chains
• paddle is outward in open form
and rotates inward in closed
form (displacement of ~15 Å)
• this pushes down on the S4-S5
linking helix (orange) causing it
to tilt down
• this presses on the S6 helix
(blue) closing the channel
Long et al. 2007, Nature 450: 376
Ligand Gated Ion Channels
• Cys-loop family of receptors
– Pentmeric receptor complexes with a central channel
– 5 subunits are homologous to each other
• Cationic channel receptors
– Allow entry of Na+ and Ca2+ (negative charges in channel)
– Depolarizes membrane (excitory)
– Acetylcholine and Serotonin are ligands for receptors
• Anionic channel receptors
– Allow entry of Cl– and HCO3– (positive charges in channel)
– Hyperpolarizes membrane (inhibitory)
– γ-aminobutyrate (GABA) and glycine are ligands for receptors
Nicotinic Acetylcholine Receptor
• Model of acetylcholine receptor
(Torpedo electric organ)
• Based on EM data on receptor and
X-ray structure of binding protein
• Pentameric receptor a2bgd with each
subunit going the length of receptor
• each subunit has 3 domains
– Extracellular β-sheet domain with 2
acetylcholine binding sites (on a2)
– Channel domain of 4 transmembrane
α-helices (M2 helix forms channel)
– Cytoplasmic domain of α-helix
a subunits are orange; ion channel is dark blue
Sine & Engel 2006, Nature 440: 448
Ligand Binding and Channel Conformations
K1*
open
R*
θ0
closed
AR*
A
θ1
A2R*
A
AR
R
K1
K1*, K2* ~ 109
K2*
K2
θ2
A2R
θ2 / θ0 = 107
K1, K2 ~ 106
• Receptor alternates between closed and open conformations
• Acetylcholine has high affinity (nanomolar Kd) for open receptors
and low affinity (micromolar Kd) for closed receptors
• Equilibrium constant between closed and open forms, θ, is 107 times
higher for receptor with 2 ligands θ2 than for unligated receptor θ0
Acetylcholine Receptor Channel Domain
Acetylcholine Receptor Mechanism
• Acetylcholine binding ~50 Å away causes 15º rotation of M2 helix
• Channel widens from 3 Å (closed) to 8 Å (open) in diameter
• Open pore allows passage of hydrated cation (~8 Å in diameter)
Ion Channels in Neural Transmission
• Ion flow through non-gated K+ channels
polarizes membrane (– inside)
• Depolarization opens then inactivates
voltage-gated Na+ channels (+ inside)
• Depolarization opens then inactivates
voltage-gated K+ channels (– inside)
• Action potential propagation opens then
inactivates more voltage-gated Na+ and K+
channels (– to + to – inside)
• Depolarization at axon end opens voltagegated Ca2+ channels ([Ca2+] to > 1 μM)
• Ca2+ activates SNAREs causing fusion of
acetylcholine vesicle with membrane; the
neurotransmitter diffuses across synapse
Ion Channels in Neural Transmission
Post-synaptic Neuron
• Acetylcholine binds to receptor
opening ligand-gated cation
channel which depolarizes
membrane (+ inside)
• Depolarization opens voltagegated Na+ and K+ channels,
stimulating the post-synaptic
neuron
• This may result in transmission
of action potential in the postsynaptic neuron
Muscle cell
• Acetylcholine binds to receptor
opening ligand-gated cation
channel, which depolarizes
membrane (+ inside)
• Depolarization opens voltagegated Ca2+ channels raising
[Ca2+] to > 1 μm
• Elevated [Ca2+] activates actin
and myosin interaction causing
muscle contraction
• Depolarization opens voltagegated Na+ and K+ channels,
propagating action potential on
muscle cell plasma membrane
Inhibitors of Ion Channels
• Voltage-gated Na+ channels
tetrodotoxin (puffer fish)
saxitoxin (Gonyaulax)
• Voltage-gated K+ channels
dendrotoxin (black mamba snake)
• Voltage-gated Ca2+ channels
w-conotoxin (cone snail)
• Acetylcholine receptors
a-conotoxin
tubocurarine
cobratoxin
bungarotoxin (Kd = 10-15)