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Potassium Channels Roger Thompson BSC5936 Membrane Biophysics Spring 2005 Florida State University Evolution of the superfamily of voltage-gated channels. Armstrong & Hille (1998)Neuron 20: 371-380 Structure Inner & Outer membrane face Layers of aromatic amino acids Tryptophan

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potassium channels

Potassium Channels

Roger Thompson

BSC5936

Membrane Biophysics

Spring 2005

Florida State University

slide2

Evolution of the superfamily of voltage-gated channels.

Armstrong & Hille (1998)Neuron 20: 371-380

structure
Structure
  • Inner & Outer membrane face
    • Layers of aromatic amino acids
      • Tryptophan
      • Tyrosine
    • Forms cuff around pore
    • Pulls pore open like springs
more structure
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
ball and chain theory
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 20:371-380

slide7

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

slide8

Putative membrane topologies of K+ channel subunits. A) voltage-gated K+ channel. B) the KATP channel

Papazian (1999) Neuron 23: 7-10

slide9

Cross section of the P region, S5, and S6 of a K+ channel.

Armstrong & Hille (1998)Neuron 20: 371-380

types of k channels
Types of K+ Channels
  • Voltage-gated
  • Inward Rectifying
  • Ca2+ sensitive
  • ATP-sensitive
  • Na+ activated
  • Cell volume sensitive
  • Type A
  • Receptor-coupled
voltage gated
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
voltage gated con t
Voltage-gated con’t
  • Activated by depolarization
  • Present in both excitable and nonexcitable cells
  • Functions
    • Regulate resting membrane potential
    • Control of the shape and frequency of action potentials
slide13

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

slide14

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

functions of delayed rectifier k channels
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
inwardly rectifying k channel
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++
functions of inward rectifier k channels
Functions of Inward Rectifier K+ Channels
  • Maintains resting membrane potential near Ek
  • Contributes to cell excitability
  • Non-conducting at (+) membrane potentials
ca 2 sensitive k channels
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
3 types ca 2 sensitive k channels
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
atp sensitive k channels
ATP-sensitive K+ Channels
  • ATP-inhibited
  • Inwardly rectifying
  • pH sensitive
  • Tetramer of 2 TM domains
  • Functions as glucose sensor in -cells
  • Blockers include Lidocaine
na activated k channels
Na+ Activated K+ Channels
  • Voltage-insensitive
  • Blocked by Mg++ or Ba++
cell volume sensitive k channels
Cell Volume Sensitive K+ Channels
  • Activated by increased cell volume
  • Blocked by Lidocaine
type a k channels
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
receptor coupled k channels
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

additionally
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)
paper 1

Paper 1

The structure of the potassium channel: Molecular basis of K+ conduction and selectivity

Doyle et al.

(1998)

Science 280:69-77

how determined
How determined?
  • X-ray crystallography and site-directed mutagenesis
  • Data refinement to 3.2Å
findings
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
slide29

Fig. 3

Inverted teepee architecture of the tetramer.

Doyle etal., (1998)Science 280: 69-77

slide30

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 280:69-77

slide31

Fig. 3

Stereoview perpendicular to membrane. Carboxyl orientation shown in white.

Doyle etal., (1998)Science 280: 69-77

slide32

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

slide33

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

slide34

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

slide35

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

paper 2

Paper 2

Contribution of the S4 segment to gating charge in the Shaker K+ channel

Aggarwal & MacKinnon

(1996)

Neuron 16:1169-1177

hypothesis testing
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
how tested
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
slide39

Fig. 2

  • Fraction of channels bound by inhibitor (circles) and fraction blocked (triangles) at different concentrations of radiolabeledAgTX1D20C and unlabled AgTX, respectively.
  • 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 16:1169-1177

slide40

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 16:1169-1177

slide41

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

slide42

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 16:1169-1177

paper 3

Paper 3

The orientation and molecular movement of a K+ channel voltage-sensing domain

Gandhi et al.

(2003)

Neuron 40:515-525

slide44
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
how tested45
How tested?
  • By accessibility of thiol-reactive probes
    • Tetramethylrhodamine maleimide (TMRM)
    • Methanethiosulfonate (MTS) reagents
      • MTSET and MTSES
  • Disulfide scanning experiments
conclusions
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
slide47

Fig. 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.
  • Cartoon representation of the resting and activated state paddle model.

Gandhi etal., 2003 Neuron 40:515-525

slide48

Fig. 3

Disulfide bond formation between S4 and the pore region.

Gandhi etal., 2003 Neuron 40: 515-525

slide49

Fig. 4

Disulfide bond between position 355C and 422c eliminates gating current.

Gandhi etal., 2003 Neuron 40:515-525

slide50

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 40:515-525

slide51

Fig. 6

Membrane topology of the KvAP S1-S4 fragment and possible interaction surface with the pore domain.

Gandhi etal., 2003 Neuron 40:515-525

slide52

Fig. 7

Accessibility and disulfide bonding results mapped suggest an alternative to the paddle theory.

Gandhi etal., Neuron 40:515-525

slide53

The

End

check it out
Check it out.
  • www.neuro.wustl.edu/neuromuscular/mother/chan.html