Ion channels as nanopores from principles to biosensors
This presentation is the property of its rightful owner.
Sponsored Links
1 / 21

Ion Channels as Nanopores - From Principles to Biosensors PowerPoint PPT Presentation


  • 52 Views
  • Uploaded on
  • Presentation posted in: General

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

Download Presentation

Ion Channels as Nanopores - From Principles to Biosensors

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

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?


Ion channels as nanopores from principles to biosensors

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


Ion channels as nanopores from principles to biosensors

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


Ion channels as nanopores from principles to biosensors

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)


Ion channels as nanopores from principles to biosensors

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)


Ion channels as nanopores from principles to biosensors

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)


Ion channels as nanopores from principles to biosensors

+

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)


Ion channels as nanopores from principles to biosensors

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


Ion channels as nanopores from principles to biosensors

1 mm

Patch Clamp Recording

  • Electrically isolate single channel in patch of cell membrane

  • Ideal for physiological applications

  • Less well suited to technological applications


Ion channels as nanopores from principles to biosensors

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


Ion channels as nanopores from principles to biosensors

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)


Ion channels as nanopores from principles to biosensors

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…


Ion channels as nanopores from principles to biosensors

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


Ion channels as nanopores from principles to biosensors

“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


Ion channels as nanopores from principles to biosensors

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


Ion channels as nanopores from principles to biosensors

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


Ion channels as nanopores from principles to biosensors

a-Haemolysin (from S. aureus)

  • Water soluble toxin

  • Forms heptamers in membranes

  • Pore formed by 14-stranded anti-parallel b-barrel (7x b-hairpin)


Ion channels as nanopores from principles to biosensors

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,…


  • Login