1. Lecture 10: Membrane potential and ion channels
Fain ch 3 end
10/5/09

3. Telomere - protects chromosome ends
Eilzabeth Blackburn found CCCCAA sequences in Tetrahymena - a ciliated microorganism

4. Chromosomes degrade w/o telomere
Work done by Jack Szostak and Elizabeth Blackburn - telomeres are conserved across species!!

5. Telomerase adds telomeres

6. Questions How do you follow membrane potential?
What can you learn from evolutionary relationships of ion channels?

7. Example - Membrane potential in hair cells
Important systems
Auditory
Vestibular
Lateral line
Hair cell responds to mechanosensation
Bending causes electrical response

9. Bullfrog inner ear Very accessible Use the sacculus Large hair cells
Responds to head movement (slow frequency)
May respond to sound

10. Frog sacculus Maculus is sensory epithelium (location marked by | | |)
From MBL Neuroscience course manual on dissection of frog sacculus by Janet Cyr

11. Hudspeth and Corey 1977 Hair cells of inner ear (bull frog) Bundles
Kinocilium
Stereocilia - microvilli
HC = hair cell
SC = supporting cell
Arrows point to kinocillium
Hair cells are in bundles- Single true cillium - kinocillium and many microvilli called stereocillia
Take hair cells from bull frog sacculus - responds to ground vibration and maybe sound
Use

12. Hudspeth and Corey 1977
Remove otolithic membrane (OM) to reveal hair cells
Use stimulus probe (SP) to perturb hair cell
Record intracellular potential with microelectrode (ME)
Hair cells are in bundles- Single true cillium - kinocillium and many microvilli called stereocillia
Take hair cells from bull frog sacculus - responds to ground vibration and maybe sound

13. Hair cell motion Towards kinocilium Depolarize
Inside cell less negative
Away from kinocilium
Hyperpolarize
Inside cell more -
Sideways motion had no effect
Depol >> Hyperpol
This shows change in inner cell potential from the resting potential of -58 mV. This resting potential occurs at 0 um displacement. So resting potential goes from-60 mV to -52 mV or so.
How can we explain this result??
Fain fig 3.11

14. How can we explain this result?
Are channels opening or closing?
What ions are moving?

15. Cell membrane contains ion pumps and channels - create concentration gradients
Inside cell
Outside cell
Na/K ATPase
Na+
141 mM
3.3 mM
Na+
15 mM
120 mM
Na/K ATPase is ion pump which is always working
Ion channels can be specific for either Na or K (color coded) and can open or close. K+ K+

16. Pump sends Na+ out Channel lets Na+ in
Inside cell
Outside cell
Na/K ATPase
Na+ pumped out
Na+
141 mM
3.3 mM
Na+
15 mM
120 mM
Na+ flows in through open channel
So closing channel prevents Na+ coming in and pumps Na+ out --> makes inside more negative - hyperpolarize
Opening channel lets Na+ in and makes inside less negative - depolarize K+ K+

17. Pump sends K+ in Channel lets K+ out
Inside cell
Outside cell
Na/K ATPase
K+ pumped in
Na+
141 mM
3.3 mM
Na+
15 mM
120 mM
Closing channel prevents K+ from going out and will still be pumped in --> inside cell gets more + so less - depolarized
Opening channel lets K+ leave so inside more - so hyperpolarize K+ K+
K+ flows out through open channel

18. Possible mechanisms Na+ channel Motion rel kino Away Toward Cell
Hyperpol
Depol
Channel
Na+
Motion is relative to kinocillium. Experiment tells us that moving towards kinocilium depolarizes cell and away hyperpolarizes
If we had a Na channel how could we explain this?

19. Possible mechanisms Na+ channel Motion rel kino Away Toward Cell
Hyperpol
Depol
Channel
Close
Open
Na+
Pump out
Flow in
Assumes must be some channels open at rest position. These are about 20% of total (1.7mV/ 8.7mV)
So 80% of channels are open at rest and then close when cell depolarizes

20. Possible mechanisms Na+ channel K+ channel Motion rel kino Away Toward
Cell
Hyperpol
Depol
Channel
Close
Open
Na+
Pump out
Flow in
K+ channel
How would K channel explain these results?
Motion rel kino
Away
Towards
Cell
Hyperpol
Depol
Channel K+

21. Possible mechanisms Na+ channel K+ channel Motion rel kino Away Toward
Cell
Hyperpol
Depol
Channel
Close
Open
Na+
Pump out
Flow in
K+ channel
So 80% of channels are open at rest and then close when cell depolarizes
Motion rel kino
Away
Toward
Cell
Hyperpol
Depol
Channel
Open
Close K+
Flow out
Pump in

22. Which is it? K+
Na+
The Thinking Man - Rodin

23. Voltage clamping Hold cell at fixed voltage
Measure current flow across membrane
Direction
Size
No change in voltage gated channels
Fig 3.13

24. Ohm’s law V = I R Current = voltage / But conductance,g is 1/R
Voltage = current *
resistance
Current = voltage /
I = V / R
But conductance,g is 1/R
I = V g V I
More resistance something has, the less it conducts current
More conductance cell has, the more current can flow. What causes conductance in a cell???? The ion channels R

25. Cell is a resistance / conductance
Resistance and conductance depend on how many channels are open
Measure current to learn about conductance
If more channels are open, more current will flow
This will tell us if channels are opening or closing in response to stimulation
Fig 3.13

26. Voltage clamping Current flow
Erev is potential at which no current flows
Potential which balances ion concentration gradient
Vm is membrane potential during stimulation
Current depends both on electrophoretic force of membrane potential and the diffusive force of concetration differences
Erev is for cell at rest with no stimulation
If hold Vm-Erev fixed, then current tells us directly about the conductance and hence about what ion channels are doing!

27. Calculate Erev for hair cells equally permeable to Na+ and K+
Outside cell
Inside cell
Na+
141 mM
3.3 mM
Na+
15 mM
120 mM
Na/K ATPase K+ K+
Essentially, total ion concentration outside and inside are the same so no gradient so no potential
=1

28. For hair cells, because Erev~0
Ion current is proportional to conductance
As stimulate hair cell, conductance changes
Voltage gated current is prop to conductance
Conductance tells us about number of ion channels opened * the conductance of each channel

29. Current flow direction
Displace toward kinocillium
Depolarization
Vm positive, current is positive
Current flows out
Vm negative, current is negative
Current flows in Vm
Displace towards kinocillium: Vm is relative to Erev so Vm positive is not a positive membrane potential but less negative than Erev.
At positive Vm, current is positive (outward)
At negative Vm, current is negative (inward)
Fig 3.14

30. Hair cell stimulus Conductance change
So movement towards kinocillium increases conductance

31. Hair cell stimulus Conductance change
So movement towards kinocillium increases conductance
Channels open
Na channels!

32. Evolution of ion channels
How are different ion channels related?
What are structural similarities?

33. K+ channel

34. Simplified 2TM channel
Roderick Mackinnon used the Streptomyces lividans channel in his Xray crystallography studies
Found it was similar to vertebrate K+ channels because both are blocked by neurotoxins
Only need 2 transmembrane TM regions and the pore region

35. K+ ion pore formed from 4 subunits

36. Ion selectivity determined by S5, S6 and pore

37. S1-S4 adds channel gating
Selectivity can be voltage gating

38. How channels are gated by voltage
Nature 423 (2003) 42-8

39. Voltage sensitive paddles - move to open and close channel
This is not only explanation. Others think that these arms rotate rather than moving up and down (Catterall 1986)

40. Large motion of S4 helix in response to charge : Arginines (+)
Used antibody binding to figure out which regions move from inside to outside membrane.
Use site directed mutagenesis to figure out which aa are part of and critical to this motion
4 positively charged argenines in S3-S4 region are key - as inside becomes less negative they move up and towards extracellular side

42. Family of ion channels Label Ion : K, Na, Ca How channel is gated:
voltage Ca
Voltage gated K, Kv
Voltage gated Cav and Nav
TRP and TPC - transient receptor potential channels
Calcium gated K
Cyclic nucleotide gated CNG and hyperpolarization activated cyclic nucleotide gated channels (HCN) - binding site which modifies movement of S6
Kir - inwardly rectifying
K2P - two pore motif

43. Root is likely the 2TM channels
All bacteria have 2TM and some have 6 TM so 2TM is primitive

44. TM channels Verts+inverts Bacteria group C Verts+inverts
Go from simplest 2TM channels to more complex.
Bacteria group B
Bacteria group A

45. Gain of S1-S4 enables voltage gating
Verts+inverts
Bacteria group C
Verts+inverts
Gain of S1-S4 allows voltage regulation
Bacteria group B
Bacteria group A

46. Some species have 4x6TM regions
Bacteria only have single 6TM gene - no 4x6TM genes
May have gene duplication + fusion to create these

47. Voltage gated sodium channel Result of gene duplication and fusion

48. Multimeric channels Verts Na+ Jelly, cnidarians, inverts Verts+inverts
Find Ca (but not Na) channels with 4x6TM in yeast
Find Na channels with 4x6TM in jellyfish, cnidarians, etc but not worm
5 of 10 Na 4x6TM are in a cluster. Others have spread to other chromosomes in parallel with hox genes - suggests result of whole genome dup
Ca+
Yeast
Na+
Bacteria

49. Phylogenies of different channels
What would be difficult about building a tree comprised of these kinds of genes?
Which region of the Nav channel is homologous with the 6TM channel on right??

50. CNG channels are important for vision and smell
CNG acquired ligand binding specificity to make them sensitive to cyclic nucleotides
Binding occurs in C terminus and is thought to exert a torque on S6 which opens channel

51. Ion channel summary Structure and function reasonably well understood
Domain and gene duplications followed by fusions played role
Diversity of ways to gate channels

52. Crystal structure of Kv channel in open state
This does not suggest S4 moves as large paddles. Stay in TM region. So some controversy to resolve!