Transcript Missy Cavallin September 14, 2007

Potassium Channels
Missy Cavallin
September 14, 2007
Doyle et al. (1998) Science 280:69-77
Outline

Types of K+ channels

Voltage-gated


Functional roles

Nomenclature

Structure

Activation

Inactivation
Assigned Experimental Papers (structure,
voltage sensor, inactivation)
Types of
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+
K
Channels
Inward Rectifying
Ca+2 sensitive
ATP-sensitive
Na+ activated
Cell volume sensitive
Type A
Receptor-coupled
Voltage-gated
Armstrong and Hille (1998) Neuron 20:371-380
Inward Rectifying (KIR)

2 transmembrane regions (M1 and M2)

4 subunits form pore

P region between M1 and M2



Regulated by concentration of extracellular
potassium
Activity dependent on interactions with
phosphatidylinositol 4,5-bisphosphate (PIP2)
Blocked by external Ba+
Functions of KIR

Maintain membrane resting potential near EK


non-conducting at positive membrane potentials
Contributes to cell excitability
+2
Ca
sensitive

4 protein subunits

Selectivity filter on external surface

RCK domains act as gate


2 Ca+2 ions bind to RCK to regulate gate
3 types of channels

High conductance (BK): 100-220 pS

Intermediate conductance (IK): 20-85 pS

Small conductance (SK): 2-20 pS
Functions of
+2
Ca
sensitive

Generate membrane potential oscillations

Afterhyperpolarization
ATP-sensitive

2 Transmembrane regions

4 subunits

Inhibited by ATP

Inwardly rectifying

Not voltage dependent
Functions of ATP-sensitive

Couples K+ conductance to cell metabolic state


Responds to metabolic changes: e.g. senses
glucose concentration in -cells
Senses intracellular nucleotide concentrations
+
Na
activated

Voltage-insensitive

Blocked by Mg+2 and Ba+2
Cell volume sensitive

Activated by increase in cell volume

Blocked by quinidine, lidocaine, cetiedil
Type A


Tetramer of -subunits and intracellular subunits
Rapid activation and inactivation


Inactivation may involve -subunits
Possible role in delaying spikes by regulation of
fast phase of action potentials
Receptor-coupled


Muscarinic-inactivated

Slow activation

Non-inactivating

Non-rectifying
Atrial muscarinic-activated

Inward rectifying
Voltage-gated channels: Function


Regulate resting membrane potential toward EK
Control shape and frequency of action
potentials





Keep fast action potentials short
Terminate intense periods of activity
Time interspike intervals
Lower the effectiveness of excitatory inputs on
a cell when open
Delayed rectifier type expressed in axons

Delayed activation and slow inactivation (shape
action potential)
Nomenclature of voltagegated K+ channels
Gutman et al. (2005) Pharmacol Rev 57:473-508
Voltage-gated: Structure


6 transmembrane
(TM) regions (S1-S6)
Principal  subunits



Homo- or
heterotetramers
S5 & S6 of each
subunit surround pore
Auxiliary  subunits

Regulate channel
activity
Gulbis et al (2000) Science 289:123-127
More Structure

S4 segment has multiple positively charged
amino acids (arginine or lysine)


P loop between S5 & S6 (selectivity filter)


voltage sensor
G-Y-G sequence required for K+ selectivity
T1 domain: tetramerization domain; connects 
subunits to  subunits
+
K
movement through channel


K+ attracted to
negative charge of
helices near
selectivity filter
K+ becomes hydrated
before exiting channel
Doyle et al. (1998) Science 280:69-77
Voltage-gated: Activation



Activated by depolarization
Voltage sensor (S4)
Translocation of charges on S4



Helical screw
Lateral movement of crossed helices
Rocking motion at interface of 2 domains
Voltage-gated: Inactivation

N-type: ball-and-chain



Amino acids at N-terminus occlude intracellular
channel pore
Rapid inactivation
C-type


Conformational changes at selectivity filter or
extracellular entrance to channel
Slow inactivation
Kurata and Fedida (2006) Progress in Biophysics and Molecular Biology 92:185-208
Rasmusson et al (1998) Circ. Res. 82:739-750
Paper 1
Crystal Structure of a Mammalian VoltageDependent Shaker Family K+ Channel
Stephen B. Long, Ernest B. Campbell, and
Roderick MacKinnon
Science (2005) 309:897-903
Methods



Kv1.2 with 2 subunit from rat brain expressed
in yeast
X-ray crystallography
2.9 Å resolution
Figure 1 A) Electron density map = blue mesh;
final model trace = yellow B) Crystal lattice structure of channel:
transmembrane segments of  subunit = red;  subunit + T1 = blue;
unit cell = black box
Kv1.2 structural features



4-fold symmetry (tetrad axis of unit cell)
Dimensions of tetramer: ~135 Å x 95 Å x 95 Å
Length of transmembrane segments: ~ 30 Å
(approximately thickness of lipid bilayer)
Figure 2 A) Side view of
ribbon model of channel with
4 subunits of unique color.
NADP+ cofactor = black sticks
B) 1 subunit from panel A
illustrating S1-S6 as well as
N- and C-termini C) Looking
into pore from extracellular
side to show interactions of 4
subunits.
Figure 3 A) Side view of 2 subunits of three voltage-gated K
channels: Kv1.2 = red; KcsA = gray; KvAP = blue; outer =
S5 of Kv1.2; inner = S6 of Kv1.2 B) Top view into pore
Note the overlap of the structures (conserved).
Paper 2
How Does a Voltage Sensor Interact with a Lipid
Bilayer? Simulations of a Potassium Channel
Domain
Zara A. Sands and Mark S.P. Sansom
Structure (2007) 15:235-244
How is a positively charged S4 helix able to
stably span the lipid bilayer?

Used molecular dynamics simulations to test
the interactions of the voltage sensor domain of
KvAP (archaebacterial) with an artificial
phosphatidylcholine (PC) bilayer or with a more
natural PC + phosphatidylglycerol (PG) bilayer


Computer modeling
Extended time (50 ns) vs. other models (20 ns) to
increase sampling
Results


Penetration of water into center of voltage
sensor and bilayer
Local deformation of bilayer due to interactions
of lipid head groups with arginine side chains of
S4 subunit


Electrostatic field is focused at center of bilayer
Numerous hydrogen bonds between phosphate
head groups of lipids with arginine residues of
S4 subunit
Figure 1 Lipids interact strongly
with and are drawn into voltage
sensor causing changes in PC
bilayer conformation. A) Voltage
sensor shown as ribbon (S1-3 =
gray; S4 = magenta); lipid
phosphate head groups colored
based on z coordinate (red =
extracellular; blue = intracellular).
Arrow shows phosphate that is
pulled away from surface. B)
Arginines on S4 interactions with
lipid phosphate groups.
Figure 2 Shows lipid bilayer compression when voltage
sensor present vs. respected control bilayers without
proteins. (distance between upper and lower phosphate
atoms)
Figure 3 There are more
hydrogen bonds formed in
PC bilayer. Although both
bilayers have hydrogen
bonds between S4 arginines
and lipid phosphate groups
or water.
Figure 4 A and C) red =
water; blue = lipid B) blue
= cationic side chain; red
= anionic side chain
PC/PG overall has more
hydrogen bonds.
Increased hydrogen
bonds at termini may
stabilize charged S4 in
bilayer.
Lack of water to
hydrogen bonds at R133
indicates prevention of
water penetration.
Figure 6 A) Water in cavities of of voltage sensor but not at
constriction point. B) Model of average pore radius. C)
Electrostatic potential distribution (± 120 mV). Note the focus
of electrical potential around salt bridge at constriction point.
Conclusions


Hydrogen bonding may help to stabilize voltage
sensor in membrane
Compression of bilayer can decrease the
distance necessary for the movement of C-type
inactivation of potassium channels
Paper 3
Slow Inactivation in Voltage Gated Potassium
Channels is Insensitive to the Binding of Pore
Occluding Peptide Toxins
Carolina Oliva, Vivian Gonzalez, and David
Naranjo
Biophysics Journal (2005) 89: 1009-1019
Toxin binding to K channels and
Slow (C-type) Inactivation



Sensitive to K+ or tetraethylammonium (TEA) in
pore
Mutations of external vestibule alter both
processes
Does toxin binding interfere with C-type
inactivation?
Figure 1 B) Amino acids important for toxin binding (left) and
slow inactivation (right) overlap. red = scorpion toxin; yellow =
conotoxin; orange = both; blue = slow inactivation
Methods

Oocytes



Whole cell recording
Outside-out patch recording
Toxins


Conotoxin (k-PVIIA)
Charybdotoxin (CTX)
Results


Rate of inactivation and recovery are toxin
insensitive
Inactivation does not alter toxin binding site
Figure 2 Whole cell voltage
clamp. A) Conotoxin (thick
trace) decrease current
amplitude and causes slight
delay in activation. B) Time
course of ratio of currents in
panel A. Toxin binding (and
current ratio) reached steady
state within recording interval.
Figure 3 and Table 1 show that the inactivation kinetics are
not affected by conotoxin in spite of change in current
amplitude. Therefore, conotoxin binding is independent of
slow inactivation. Similar results were shown for CTX (Fig.
6). There are differences between whole cell vs. outside
out patches, but not with regards to toxin effects.
Figure 5 A) Conotoxin decreases current amplitude in
response to voltage steps. B) IV and GV relationships.
Voltage shift does not change in the presence of toxin
(bottom). DV = -18 mV for no toxin; DV = -21 mV with toxin
Conclusions


Inactivation kinetics were not affected by toxin
binding
Bound toxin does not hinder conformation
change involved with slow inactivation