1. Welcome to 725 nCellular and Molecular Neuroscience nChris Elliott & Sean Sweeney nAim: describe the cellular workings of the CNS u in health and disease F Neurons F Glia F Blood vessels nSee http://biolpc22.york.ac.uk/725http://biolpc22.york.ac.uk/725

2. Neurons nWhy are neurons so interesting ? u Fast signalling u Specific connections u Long distances nKey features: u Need glia u Ion channels u Synaptic transmission

3. Glia nAbout 100 times more glial cells than neurons nSupport neurons

4. Human CNS

5. Revision – cell shape nAxon nDendrites nSoma

6. Channel distribution nNon-uniform u Different in cell body and axon/dendrites u Myelinated axons – Na channels at node of Ranvier K orange; Na red

7. Na channel is anchored

8. Node of Ranvier nHow does it develop? Cell adhesion molecule recruits ankyrin Caspr (axon) + cell adhesion molecule

9. Node of Ranvier nHow does it develop? Caspr in axon, linked to cell adhesion molecule in Schwann Cam x 3

10. Summary so far nNeuronal organisation is complex u Cell geometry u Channel distribution nSignalling by cell-cell interaction important for organistion

11. Revision - electrics nCurrent is rate at which ions flow u Measure in ions/sec or Amps nVoltage is driving force nResistance = V/I nConductance = I/V u More current flowing means a bigger hole to flow through u Measure in Siemens S (pS)

12. Revision – voltage clamp nAim: to separate capacitance current (I C ) from ionic current u I C only flows when the voltage is changing u Use ion substitution or pharmacological blockers to identify ionic currents

13. Not all APs are equal nAction potentials in u Myelinated u Unmyelinated u Cell bodies u Dendrites u Snails nNote differences in time scale!

14. Not all APs are equal nAction potentials in u Myelinated u Unmyelinated u Cell bodies u Dendrites u Snails nMammals are different to amphibians

15. Not all APs are equal nMammals have many less K channels nAP depends on inactivation of Na current to end

16. Many types of channels nIon channels for Na, K, Ca, Cl, etc nSubtypes for each ion may have different characteristics u Here 3 K channels F Maintained F Transient F Off transient

17. VC- refractory period nTwo pulse experiment u K-current blocked u Na current only

18. VC- gating current nIf Na channels are opened by voltage, then they need a voltage sensor nMeasure the current when Na and K are blocked K current blocked Na and K current blocked Na current (subtraction)

19. Is it really gating current? nTwo pulse experiment u K-current blocked u Na current only Plot initial Na vs gating current

20. Is it really gating current? nMostly ? nCorresponds to movement of about 3 ionic charges nAlso measure using asymmetry of positive and negative pulses, so may be called asymmetry current Na current “Gating current”

21. Summary point nMacroscopic analysis shows: u Voltage sensitivity important in axons u Physiological diversity to reflect anatomical diversity u Implies cellular diversity

22. Revision – patch clamp nUse a small patch of membrane u Fixed voltage u Measure current

23. Summated channels nSummation of the effects of individual channels give the macroscopic result

24. Properties of channels nObey Ohm’s law nIons flow freely through open channels nChannels selective for particular ions

25. Channels vs transporters nChannels flow freely nTransporters need energy u ATP u ion gradient

26. Molecular biology n4 repeats of 6 transmembrane regions n S4 mutations affect opening n S6 line the pore

27. Mutations for disease? nMost mutations probably fatal before birth

28. Channel radiation nSimilar genes encode channels with different ionic specificity Na Ca cyclic K

29. Opening and closing? nInactivation (closing) u Ball and chain mechanism

30. Activation (opening) u Mutagenesis of +ve charged amino- acids affects voltage sensitivity + residues  Helix screw model

31. New hypothesis nRotation of charged residues in S4 may affect S5 and S6 to change diameter of the pore

32. Alternative splicing

33. RNA Editing nADARs (adenosine deaminases that act on RNA) A → I (treated as G)

34. How often in ion channels? nMultiple genes in mammals (9) nMuch alternative splicing nMany RNAi editing sites u Glu ion channels u Serotonin receptor u Potassium voltage gated channels nIn flies, u one Na channel gene u > 3 alternative spices u 10 RNAi editing sites

35. Conclusion nMicroscopic physiology and molecular studies contribute together to our understanding of channels nMechanism of opening and of closing relates to channel morphology and sequence nEvolutionary diversity and adaptation to different functions nReferences