Potassium Channels

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Potassium Channels

• Roger Thompson
• BSC5936
• Membrane Biophysics
• Spring 2005
• Florida State University

2
Evolution of the superfamily of voltage-gated
channels.
Armstrong Hille (1998)Neuron 20 371-380
3
Structure

• Inner Outer membrane face
• Layers of aromatic amino acids
• Tryptophan
• Tyrosine
• Forms cuff around pore
• Pulls pore open like springs

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More structure

• Two gating theories
• Ball and chain
• Paddle
• Selectivity filter
• A narrow region near outer face of membrane
• Contains glycine-tyrosine-glycine residues
• Is lined with carbonyl backbone
• Ions travel through in single file

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Ball and Chain Theory
When the channel is open (center), any one of the
four inactivation balls can inactivate the
channel (right). Inactivation for a Na channel
is similar, but there is a single inactivaton
ball.
Armstrong Hille (1998) Neuron 20371-380
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Paddle Theory
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Structure of voltage-gated ion channels.
Functional components (A) and peptide folding (B)
are shown diagrammatically with P regions in red
and the S4 segment in pink.
Armstrong Hille (1998)Neuron 20 371-380
8
Putative membrane topologies of K channel
subunits. A) voltage-gated K channel. B) the
KATP channel
Papazian (1999) Neuron 23 7-10
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Cross section of the P region, S5, and S6
of a K channel.
Armstrong Hille (1998)Neuron 20 371-380
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Types of K Channels

• Voltage-gated
• Inward Rectifying
• Ca2 sensitive
• ATP-sensitive
• Na activated
• Cell volume sensitive
• Type A
• Receptor-coupled

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Voltage-gated

• 6 transmembrane domains
• 4 subunits surround central pore (S5 S6 regions
  of each subunit
• Selectivity filter (P region)
• Hydrophobic sequence between last 2 TMD contains
  Gly-Tyr-Gly
• Voltage sensor (S4) has multiple positively
  charged amino acids

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Voltage-gated cont

• Activated by depolarization
• Present in both excitable and nonexcitable cells
• Functions
• Regulate resting membrane potential
• Control of the shape and frequency of action
  potentials

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Composite model of a voltage-dependent K
channel. The ? subunit is shown in red and the ?
subunit in blue.
Gulbis etal., (2000) Science 289 123-127
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KirBac1.1 structure consisting of an all
?-helical integral membrane section plus an
intracellular domain consisting mostly of ?-sheet.
Kuo etal., (2003) Science 300 1922-1926
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Functions of Delayed Rectifier K Channels

• Delayed activation slow inactivation
• Allows efficient repolarization after action
  potential
• Structure tetramer of ?-subunits ? ?subunits
• Can be blocked by
• 4-aminopyridine, Dendrotoxins, Phencyclidine,
  Phalloidin, 9-aminoacridine, Margatoxin,
  Imperator toxin, Charybdotoxin

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Inwardly Rectifying K Channel

• 2 transmembrane regions (M1 M2)
• Corresponds to S5 S6 in Kv channel
• 4 subunits surround central pore
• P region separates M1 and M2
• Non-conducting at positive membrane potentials
• Blocked by external Ba

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Functions of Inward Rectifier K Channels

• Maintains resting membrane potential near Ek
• Contributes to cell excitability
• Non-conducting at () membrane potentials

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Ca2 Sensitive K Channels

• Generate membrane potential oscillations
• 4 protein subunits
• External surface contains selective K filter
• Inner cavity accommodates a hydrated K ion
• 2 Ca2 ions binds to RCK domains interact
  regulate gate

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3 Types Ca2 Sensitive K Channels

• High conductance (BK) channels
• Gated by internal Ca2 and membrane potential
• Conductance 100 to 220 picoSiemens (pS)
• Intermediate conductance (IK) channels
• Gated only by internal Ca2
• More sensitive than BK channels
• Conductance 20 to 85 pS
• Small conductance (SK) channels
• Gated only by internal Ca2
• More sensitive than BK channels
• Conductance 2 to 20 pS

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ATP-sensitive K Channels

• ATP-inhibited
• Inwardly rectifying
• pH sensitive
• Tetramer of 2 TM domains
• Functions as glucose sensor in ?-cells
• Blockers include Lidocaine

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Na Activated K Channels

• Voltage-insensitive
• Blocked by Mg or Ba

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Cell Volume Sensitive K Channels

• Activated by increased cell volume
• Blocked by Lidocaine

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Type A K Channels

• Possible regulation of fast repolarizing phase of
  action potentials delay spiking
• Tetramer of ?-subunits intracellular ?-subunits
• ?-subunits may confer rapid inactivation
• Blockers include Phencyclidine and Dendrotoxins

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Receptor-coupled K channels

• Blockers include Ba, Bradykinin, Cs, TEA and
  Quinine
• Two types
• Muscarinic-inactivated
• Slow activation
• Non-inactivating
• Non-rectifying
• Atrial muscarinic-activated
• Inward rectifying

Muscarinic acetylcholinergic
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Additionally

• Greater tendency to allow K to flow into cell
  than to flow out
• Regulated by extracellular K concentration
• Inward rectification due mainly to internal
  magnesium block of outward current
• Dependent on interaction with phosphatidylinositol
  4,5-bisphosphate (PIP2)

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Paper 1

• The structure of the potassium channel
  Molecular basis of K conduction and selectivity
• Doyle et al.
• (1998)
• Science 28069-77

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How determined?

• X-ray crystallography and site-directed
  mutagenesis
• Data refinement to 3.2Å

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Findings

• Inverted teepee shape
• Selectivity filter diameter 12Å
• Carbonyl O2 line selectivity filter
• K is 10.000x more permeable than Na
• Both side of pore are (-) charged
• The pore is hydrophobic

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Fig. 3
Inverted teepee architecture of the tetramer.
Doyle etal., (1998)Science 280 69-77
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Fig. 3
Stereoview of a ribbon representation
illustrating the three dimensional fold of the
KcsA tetramer viewed from the extracellular side.
The four subunits are distinguished by color.
CS Streptomyces lividans
Doyle etal., (1998)Science 28069-77
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Fig. 3
Stereoview perpendicular to membrane. Carboxyl
orientation shown in white.
Doyle etal., (1998)Science 280 69-77
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Fig. 3
Ribbon representation of the tetramer as an
integral-membrane protein. Aromatic amino acids
are displayed in black.
Doyle etal., (1998)Science 280 69-77
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Fig. 7
Two mechanisms by which the K channel stabilizes
a cation in the middle of the membrane. First, a
large aqueous cavity stabilizes an ion (green) in
the otherwise hydrophobic membrane interior.
Second, oriented helices point their partial
negative charge (carboxyl end, red) towards the
cavity where a cation is located.
Doyle etal., (1998)Science 280 69-77
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Fig. 4
A cutaway stereoview displaying the
solvent-accessible surface of the K channel
colored according to physical properties. Blue
high positive charge Red negative
charge Yellow hydrophobic C atoms Green K
ion positions White neutral charge
Doyle etal., (1998)Science 280 69-77
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Fig. 4
A three-dimensional stick model representation of
the minimum radial distance from the center of
the channel pore to the nearest van der Waals
protein contact.
Doyle etal., (1998)Science 280 69-77
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Paper 2

• Contribution of the S4 segment to gating charge
  in the Shaker K channel
• Aggarwal MacKinnon
• (1996)
• Neuron 161169-1177

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Hypothesis testing

• Used Shaker K channels expressed in Xenopus
  oocytes
• Neutralized positive charges in the S4 segment
• Measured reduction in gating charge.
• This reduction would represent the contribution
  of the positively charged residue to the gating
  charge of the channel

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How tested?

• Mutagenesis to create AgTX combined with
  tritiated N-ethylmaleimide
• Based on extinction coefficient of nontritiated
  NEM-labeled AgTX, the specific activity of
  radiolabeled toxin was determined by measuring
  disintegrations per min as a function of toxin
  concentration

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Fig. 2

1. Fraction of channels bound by inhibitor (circles)
   and fraction blocked (triangles) at different
   concentrations of radiolabeledAgTX1D20C and
   unlabled AgTX, respectively.
2. Tritiated AgTX1D20C binding data for oocytes
   expressing Shaker K channels. U uninjected, I
   oocytes expressing channels, C 40X conc.
   Injection of unlabeled AgTX

Aggarwal MacKinnon (1996) Neuron 161169-1177
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Fig. 3
B) Repolarization-induced currents integrated
over time to give total charge, and plotted as a
function of pulse potential (Q-V). Dashed line
linear capacitance of the cell and voltage clamp
system nonlinear component gating charge
movement D) Q-V plot of repolarization-induced
current is linear.
Aggarwal MacKinnon (1996) Neuron 161169-1177
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Fig. 3
E) Correlation plot mapping total gating charge
(q) in electron charge units as a function of
total channel number (n) for several oocytes
expressing Shaker K channels. The line
corresponds to a linear regression fit using the
method of least squares with a 95 confidence
interval.
Aggarwal MacKinnon (1996) Neuron 16 1169-1177
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Fig. 7
Gating charge movement (q/n) for the wild-type
Shaker K channel and charge-neutralizing (B) and
charge-conserving (C) S4 mutations as a function
of pulse potential. D) A model of Shaker K
gating in which the S4 segment undergoes a change
in secondary structure.
Aggarwal MacKinnon (1996) Neuron 161169-1177
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Paper 3

• The orientation and molecular movement of a K
  channel voltage-sensing domain
• Gandhi et al.
• (2003)
• Neuron 40515-525

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What?

• New model of voltage sensing domain
• where S4 lies in the lipid, at the channel
  periphery
• and moves through the membrane
• as a unit with a portion of S3

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How tested?

• By accessibility of thiol-reactive probes
• Tetramethylrhodamine maleimide (TMRM)
• Methanethiosulfonate (MTS) reagents
• MTSET and MTSES
• Disulfide scanning experiments

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Conclusions

• found that the S1-S3 helices have one end that is
  externally exposed
• that S3 does not undergo a transmembrane motion
• and S4 lies in close apposition to the pore
  domain in the resting and activated state

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Fig. 1

1. KvAP structure and the activated state paddle
   model labeled to indicate several sites examined
   for S-S bonds formation between S4 and the pore
   domain and for state-dependent accessibility to
   MTS reagents.
2. Cartoon representation of the resting and
   activated state paddle model.

Gandhi etal., 2003 Neuron 40515-525
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Fig. 3
Disulfide bond formation between S4 and the pore
region.
Gandhi etal., 2003 Neuron 40 515-525
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Fig. 4
Disulfide bond between position 355C and 422c
eliminates gating current.
Gandhi etal., 2003 Neuron 40515-525
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Fig. 5
Disulfide bonding to S5 depends on the location
of the S3-S4/S4 cysteine and on the gating state.
Gandhi etal., 2003 Neuron 40515-525
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Fig. 6
Membrane topology of the KvAP S1-S4 fragment and
possible interaction surface with the pore domain.
Gandhi etal., 2003 Neuron 40515-525
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Fig. 7
Accessibility and disulfide bonding results
mapped suggest an alternative to the paddle
theory.
Gandhi etal., Neuron 40515-525
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The

• End

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• www.neuro.wustl.edu/neuromuscular/mother/chan.html