ion channels as nanopores from principles to biosensors
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Signal IN. Signal OUT. Sensor. Ion Channels as Nanopores - From Principles to Biosensors. What to detect? Why pores ? Why bio ?. Overview. Today: Principles and proteins. Review of basic properties of channels & pores Single channel measurements – technologies

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Presentation Transcript
ion channels as nanopores from principles to biosensors
Signal IN

Signal OUT

Sensor

Ion Channels as Nanopores - From Principles to Biosensors
  • What to detect?
  • Why pores?
  • Why bio?
slide2
Overview

Today: Principles and proteins

  • Review of basic properties of channels & pores
  • Single channel measurements – technologies
  • Conductance, selectivity, block…
  • Model channels for bionanotechnology:

gramicidin, alamethicin, a-haemolysin

Tomorrow: Applications

slide3
Biological Roles of Ion Channels
  • Ion channels are found in all cells but are of especial importance in neurones
  • Opening/closing of channels with different ion selectivities give changes in DV across cell membranes
  • Channels are important in propagation of action potentials and in synapses
slide4
2x
  • Dimensions
  • pore radius ca. 0.2 nm (2Å)
  • pore length ca. 3 nm (30Å)

Molecular Picture of a Transbilayer Pore

K channel in a membrane

10x

  • Can we design simpler systems?
  • Can we exploit the complexities of ‘real’ channels?

Water radius ca. 0.14 nm (1.4Å)

slide5
EC

F

C

G

IC

“Real” Channels: Too Complex for Bionano?…

F

C

G

KcsA – a bacterial K+ channel

Filter & gate regions govern activity

  • Complex biological functions
  • Large scale expression is time-consuming
atomic scale effects
Atomic scale effects
  • Example: Water in hydrophobic model pores (computer simulations)
slide7
Basic Aims…
  • Explore channel structure/function at the single molecule level: provides a more fine-grained biophysical model than experiments on populations of molecules
  • Exploit our biological understanding: develop a chemical biology of channels
  • Develop channel-based nanotechnologies: e.g. novel biosensors. Exploit
    • Small dimensions, hence single-molecule detection
    • Atomic scale effects (e.g. specific interactions, hydrophobic effects)
slide8
+

Single Channel Measurements …

amplifier

DV

can resolve small (ca. 1 pA) ionic currents with good (ca. 10 msec) time resolution

permeation (107 ions s-1)

slide9
Channels…Physiological Measurements
  • Single channel recording – 5 pA = 3 x 107 ions s-1
  • I/V curve – ohmic conductance vs. rectification
  • Gating analysis – what causes a channel to open/close?

Ohmic

rectification

slide10
1 mm

Patch Clamp Recording

  • Electrically isolate single channel in patch of cell membrane
  • Ideal for physiological applications
  • Less well suited to technological applications
slide11
DV

amplifier

bilayer between two electrolyte-containing chambers

Planar Bilayer

  • Electrically isolate small patch of bilayer
  • Can insert peptides or proteins into bilayer
  • Easy access to solutions on both sides
slide12
Channels…Concepts
  • Conductance…ca. 107 ions s-1… pore (P)
  • Selectivity…M+ vs. X-; Na+ vs. K+ … filter (F)
  • Block… ions; drugs; toxins
  • Gating… voltage-gated vs. ligand gated … gate (G)

open

closed

F

gating (~1 ms)

P

G

permeation (10 to 100 ns)

slide13
Information Encoded in Single Channel Currents…

current (pA)

C

D

A

B

time (ms)

A – ‘wild type’ current

B – reduced conductance (i.e. fewer ions sec-1)

C – open channel block (i.e. interruptions to ion flow)

D – incomplete channel block

By measuring such changes we can sense events in a channel…

slide14
Kinetics of Channel Block

channel C

tU

+ blocker ‘B’

tB

  • Average over many channel events & different [B] values
  • C + B  CB:

mean(tB) = kOFF-1; mean(tU) = (1+kON[B])-1 ;

  • KD = kOFF/kON
  • For charged blockers … KD depends on DV
slide15
“Simple” Channels for Bionanotech…
  • Gramicidin – a simple peptide that forms dimeric cation selective channels
  • Alamethicin – an amphipathic a -helical peptide that forms voltage-activated channels
  • a-Haemolysin – a bacterial protein toxin that forms large channels open to covalent and non-covalent modification
slide16
Gramicidin – A Simple Model Channel
  • Peptide – smaller & simpler than a channel protein
  • Channel properties – cation selective: M+ & H+ conductance
  • Structure – two ‘open’ helices (unusual conformation due to alternating L- and D-amino acids in sequence);
  • Single file of water (+ ion) inside the central pore
  • Structural polymorphism…unexpected complexities!
  • helical dimer forms transiently
slide17
DV

x N

surface binding

helix insertion

bundle formation

Alamethicin

Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu-Gln-Phol

(Aib = a-aminoisobutyric acid – strongly promotes a-helix formation)

apolar

polar

slide18
a-Haemolysin (from S. aureus)
  • Water soluble toxin
  • Forms heptamers in membranes
  • Pore formed by 14-stranded anti-parallel b-barrel (7x b-hairpin)
slide19
b-Barrel Pores

PhoE trimer – aromatic (Trp & Tyr) bands

PhoE trimer – pore lining basics (blue)

  • Well understood – structure; function; mechanism
  • Relatively easy to over express
  • Physically robust
take home message
Take home message

Nature uses nano pores as sensors with high

  • Sensitivity
  • Specificity

…exploiting atomic scale effects.

For “engineering” applications: simple pores

Same physical/chemical principles apply to simple and complex pores.

tomorrow
Tomorrow

Non-science:

  • observers (observing me)
  • mini questionaire (feedback for me)

“Please write down up to three points that you felt were most important in the lecture.”

“Please write down up to three points that were unclear, should be clarified, or simply better explained.”

Making sensors from gramicidin, alamethicin, HL,…

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