Ion Channels as Nanopores - From Principles to Biosensors

1. Signal IN Signal OUT Sensor Ion Channels as Nanopores - From Principles to Biosensors • What to detect? • Why pores? • Why bio?

2. 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

3. 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

4. 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)

5. 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

6. Atomic scale effects • Example: Water in hydrophobic model pores (computer simulations)

7. 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)

8. + 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)

9. 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

10. 1 mm Patch Clamp Recording • Electrically isolate single channel in patch of cell membrane • Ideal for physiological applications • Less well suited to technological applications

11. 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

12. 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)

13. 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…

14. 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

15. “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

16. 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

17. 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

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

19. 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

20. 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.

21. 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,…