Modulation of ion channels:
Looking for interactions right in the neighborhood.
M.L. Molina
M.L. Renart
I. Triano
E. Montoya
J.L. Ayala
A.M. Fernández
J.A. Encinar
J.A. Poveda
J.M. González-Ros
G. Fernández
F. Gavilanes
Xorret del Catí, 2009
The point:
Interaction with neighboring molecules are of the
essence to understand the behavior of ion channels
as we approach physiological conditions
Membrane lipids are obviously in the neighborhood but …,
why should they be considered potential interaction partners…?
A) Anionic phospholipids seemingly
needed for activity (Heginbothan et al.,
1998)
C) Reconstitution into lipid
vesicles favors “clusters”
(Molina et al., 2004).
B) KcsA crystal contains lipid (Valiyaveetil et al.,
2002), tentatively identified as PG, noncovalently bound to non-annular protein sites.
“Simple” experimental conditions: Fluorescence monitoring of KcsA
thermal stability in detergent solution (1mM DDM) and 100 mM NaCl.
λex=280 nm, λem=340 nm
50
60
70
80
90
T e m p e ra tu re , ºC
Phospholipids added to form mixed lipid-detergent micelles:
How do they affect the protein thermal stability?
- DOPE (18:1)
Zwitterionics
(Annular sites)
Anionics
(Non-annular sites)
- DOPC (18:1)
- DOPG (18:1)
- DMPG (14:0)
- Egg PG
- DOPA (18:1)
- Egg PA
All phospholipids stabilize the protein, but the anionic ones,
binding to non-annular protein sites, are much more effective
DOPG
DOPE
[lipid]
0 mM
0,02 mM
0,06 mM
zwitterionics
PGs
PAs
Alkyl sulfates can do it too …
… as long as the chain length is sufficiently long
Sodium Dodecyl Sulfate (SDS)
[alkyl sulfate]=0.25 mM
● Control
○ Hexyl
▼ Octyl
Δ Decyl
■ Dodecyl
□ Tetradecyl
Interaction between KcsA and non-annular phospholipids:
A model based on chrystallographic data and local docking
DOPG
DOPC
SDS
First take-home message:
“The stability of a membrane protein can be greatly dependent on its
interaction with neighboring, specific lipids at specific protein sites”
Clustering and coupled gating in KcsA:
diversity based on a 27 pS single channel conductance.
5 μM
ML Molina, FN Barrera, AM Fernandez, ML Renart, JA Poveda, JA Encinar, G Riquelme and JM GonzalezRos: “Clustering and coupled gating modulate the activity in KcsA, a potassium channel model”. J. Biol.
Chem. 281, 18837-18848 (2006).
Clustering and coupled gating in other systems:
More frequent than initially thought
•
Ryanodine receptors alone (Marx et al, 1998 and 2001; Yin et al, 2005), as well as together
with dihydropyridin receptors (Protasi, 2002) or Kv2.1 potassium channels (Antonucci et al,
2001; Misonou et al, 2004 and 2005).
•
Kir 4.1 potassium channels (Horio et al, 1997; Butt and Kalsi, 2006).
•
Cystic fibrosis transmembrane conductance regulator chloride channels (Krouse and Wine,
2001).
•
Nicotinic acetylcholine receptors (Schindler et al.,1984; Keleshian et al., 2000; Huh and Fuhrer,
2002).
•
Many members of the ATP-gated channel and the G-protein-coupled receptor families (see for
instance Rios et al, 2001; Herrick-Davis, 2004,2005 and 2006; Fujiwara and Kubo, 2004),
including rhodopsin (Mansoor, PNAS, 2006).
•
Sodium channels in non-Hodgkin & Huxley behaviour in mammalian neurons (Naundorf et al,
2006; Baranauskas and Martina, 2006).
“Clustered assemblies in which the activity of one receptor influences that of its
neighbours, provide the means to convert conformational changes from a single origin
into intermolecular allosteric behaviour” (Bray et al., Annu. Rev. Biophys. Bioeng., 2004).
Anionic phospholipids uncouple channel gating
Few HOPs are present in high anionic lipid …
Asolectin (~12% PA)
… and those remaining are
quite unstable.
Egg PG 25%
Anionic phospholipids disassemble channel clusters…
SDS-PAGE
of partly crosslinked samples
Confocal microscopy:
Fluorescent KcsA on supported bilayers
5% DOPG
100% DOPG
10 μm
Therefore, anionic lipids uncouple gating and disassemble KcsA
clusters, but … how is it done?
Global, unrestricted docking reveals that “non annular” lipid-protein interaction
sites partly coincide with protein-protein interaction sites involved in clustering
Sitio anular
Sitio no anular
Designing “anti-cluster” peptides
Front view
Side view
Upper view
“Anti-cluster” peptides confirm the hypothesis.
Uncoupling of channel gating
Disassembly of clusters
Control
Peptide 6 (1 μM)
HOP
LOP
LOP
+200 mV
“Anti-cluster” peptides confirm the predictions from the docking model by
uncoupling KcsA channels structural and functionally.
Key mutants of KcsA also confirm
the hypothesis from the docking model.
KcsA W87A
Second take-home message:
- “Channel clustering and coupled gating, two main processes in the
modulation of KcsA, depend strongly on the interaction with neighboring
lipids”.
- “There is an interference between lipid-protein and protein-protein
interactions”.
KcsA crystal structure:
providing insights into how ion channels work
Before …. and after
Efficient permeation
(~108 ions/s)
Selectivity:
Snug-fit hypothesis: proper interaction
with a rigid SF
K+ atomic radius: 1.33 Å
1) Binding to S1 to S4
2) electrostatic repulsion
between K+ ions
Na+ atomic radius: 0.95 Å
Global structural consequences associated to ion selectivity
Changes in tertiary structure
Changes in secondary structure
KCl
100 mM
KCl
NaCl
DDM solubilized KcsA
NaCl
100 mM
Asolectin reconstituted KcsA
K+ and Na+ compete for the binding to the channel
[K+]: 5 mM
[Na+]: 5 mM
NaCl
NaCl
KCl
KCl
Kd KcsA●K:
8 ± 2 mM
Kd KcsA●Na: 190 ± 80 mM
Renart ML, Barrera FN, Molina ML, Encinar JA, Poveda JA, Fernández AM, Gómez J, González-Ros JM. “Effects of conducting and
blocking ions on the structure and stability of the potassium channel KcsA.” (2006) J Biol Chem 281(40):29905-15.
Zhou et al., Nature, 2001.
Morais-Cabral et al., Nature, 2001.
Lockless et al., PloS Biology, 2007.
KCl 3 mM
NaCl 200 mM
KCl > 20 mM
Thermal denaturation at increasing Na+ or K+ concentration
(no competition conditions)
KCl-2
KCl-1
Non-conductive SF
Conductive SF
NaCl
Kd app.,
mM
KCl-1
KCl-2
NaCl
nH
0.020 ± 0.001
12.7 ± 0.6
17 ± 0.4
Non-conductive SF
KCl-1
KCl-2
NaCl
0.92 ± 0.09
1.19 ± 0.05
1.04 ± 0.04
Δ Tm
ºC
8.1 ± 0.3
42 ± 1
22.4 ± 0.8
Mutant channels: Tools to support the hypothesis
Conductive
Selectivity Filter
Non-Conductive
Selectivity Filter
M96V
WT
KCl 100 mM
NaCl 100 mM
M96V
KCl 100 mM
NaCl 100 mM
Induced-fit model
“high
affinity”
“low
affinity”
“low
“low
affinity”
affinity”
Phospholipid:
Allosteric
Effector?
Third take-home message:
“Ions are effectors (“ligand-like”) of ion channels and determine
greatly their stability and functionally-competent conformation”
The point:
Interaction with neighboring molecules are of the essence to
understand the behavior of ion channels as we approach
physiological conditions
The messages:
• The stability of a membrane protein is greatly dependent on its
interaction with neighboring, specific lipids at specific protein
sites
• Channel clustering and coupled gating, two main processes in the
modulation of KcsA, depend strongly on the interaction with
neighboring lipids. In other words, there is an interference
between lipid-protein and protein-protein interactions
• Ions are effectors (“ligand-like”) of ion channels and determine
greatly their stability and functionally-competent conformation