1. Lecture 12 : Metabotropic signaling and mechanoreceptors Fain begin ch 5 10/12/09

2. 2009 Nobel prize in Literature Writes about life in Romania under a dictatorship German minority

3. Nobel Peace prize Awarded the prize for who he is and what he hopes to accomplish Change in attitude and galvanizing the world

4. Nobel homework  Due on Wednesday but…  We are going to discuss two papers on Wed - each of you will be responsible for one figure  …so Nobel homework can be handed in Wednesday (10/14) or next Monday (10/19)

5. Signal transduction  Ionotropic Directly gate ion channel  Metabotropic Gate ion channel through a G protein and 2nd messenger

6. GPCR signaling    GDP GPCR *    + M=2nd messenger sends signal to ion channel M Receptor G protein Effector GTP

7. Mechanoreceptors Fain ch 5

8. Mechanoreception  Responds to mechanical pressure or distortion Hearing Touch Acceleration detection  Why are ionotropic mechanisms good for mechanoreception?

10. Express and patch clamp piece of membrane - record current as apply pressure

11. MscL - Mechanosensative channel, large  Large conductance  5 subunits x 2 TMs M1 faces pore M2 faces membrane Hydrophobic pore keeps water from flowing across membrane

12. Pore opens like iris M1/M2 rotate- Opens S1 helices Sukarev and Ankirin 2004 Channel responds to membrane tensions Opening enables ion flow/water to relieve osmotic pressure

13. Anishkin and Sukharev 2009

14. Gillespie and Walker 2001

15. Mechanoreception  Evolved multiple times Recruited different ion channels each time  3 means of tethering and pulling to open channel Direct Indirect through molecule Indirect through mechanosensitive protein

16. Questions 1.How do mechanosensory cells work? 2.Is there a common mechanical structure? 3.Is there a common molecular transduction mechanism? Gillespie and Walker 2001

17. Goals for mechanosensation 1.Maximize speed of signal detection 2.Maximize sensitivity of response

18. Paramecium sense of touch  Differential response:  If touch front, reverses direction, turn and go another way  If touch back, swims faster

19. Paramecium sense of touch  Touch front (anterior) Depolarization Inflow of Ca +2 If > 10 -6 M causes cilia to reverse direction  Touch back Hyperpolarization Outflow of K + Cilia beat faster  Touch middle Nothing happens  Difficult to figure out what the genes are Eckert 1972

20. Genomics of Paramecium sensory receptors??? 72 Mb 40,000 genes

21. C. elegans  The worm 959 cells 302 neurons  Many methods available for studying pathways  C. briggsae is closely related worm

22. Forward genetics approach  Find or make mutants with particular phenotype Chemical mutagen ENU N-ethyl N-nitrosourea Transposons Normal Mutant

23. Forward genetics approach  Find or make mutants with particular phenotype  Discover which gene is broken and so critical for phenotype Li..Xu 2006 Normal Mutant

24. Li et al 2006 : Sixth sense in worms One of papers we will read for Wed

25. Reverse genetics  Have the genes and need to figure out what they do  Make GFP reporters See where gene is expressed  Make knock-outs See what happens if gene is removed

26. C. elegans methods are worked out

27. C. elegans  Touch front Worm moves backward  Touch back Worm moves forward  Touch middle No effect  C. briggsae is closely related worm

28. Wormbook

29. C. elegans sense of touch  6 mechanosensory neurons Anterior - AVM, ALML, ALMR Posterior - PLML, PLMR  Can ablate cells and see if sense is affected Front

30. Wormbase describes all 959 cells…

31. ..including its cell lineage But not what it’s name means!!!

32. C. elegans sense of touch  AVM - anterior ventral microtubule cell  ALML/R - anterior lateral microtubule cell Left/right  P = posterior  Microtubule cell - filled with tubulins Front

33. Skin connected to cytoskeleton by receptor MEC - mechanosensory proteins identified from mutants Ion channel Tubulins

34. Mechanoreceptor

35. Worm touch  Touch causes ion channel to open and cell to depolarize  Uses many proteins which are all necessary Can make knockins or outs of each gene and figure out how mechanoreceptor works But hard to record from neurons  Ion channel is similar to epithelium Na channel Humans have a dozen of these - likely important in mechanosensation

36. Crayfish  Large enough for intracellular recording Genetics are difficult  Abdominal stretch receptors MRO = muscle receptor organ

37. Stretch receptor

38. Crayfish mechanoreceptor  Intracellular recording from cell attached to muscle  Stretch muscle and record

39. Depolarize and generate action potentials MRO 1 - continuous response during stretch - slow adapt MRO 2 - respond only at first - fast adapt

40. Another differences in MRO 1 and MRO 2 adaptation  Also differences under voltage clamped conditions Rate of fiber relaxation Greater adaptation in MRO2

41. Crayfish stretch receptors  May be directly responding to membrane stretch  To test this, pull off patch and apply pressure to see response

42. Two kinds of channels  Stretch activated - independent of voltage, in dendrites, many SA channels  Rectifying SA depend on voltage, in cell body, few RSA channels SA RSA

43. Cray fish genomics?  Genome size 5-6 Gb

44. Insect mechanoreceptors  Type I Bipolar Cilium at base of outer segment Extracellular structures Bathed in high K + medium secreted by supporting cells  Type II Multipolar - many dendrites Associate with internal organs or skin No supporting cells

45. Type I - Hair plate sensillum  Outer segment Connects to base of bristle Microtubules surround by extracellular matrix

46. Hair cell sensillum  Bristle motion causes cuticle to push on cap  Cap pushes on tubular body Depolarization Likely cation channels open

47. Type I - Campaniform sensillum Senses compression of cuticle

48. Type I - Scolopidial organ  Scolopale cell - supporting cell Secretes extracellullar membrane  Detect vibrations transmitted from cuticle through accessory cell Tympanal organs Johnston’s organ

49.  Located in antennae  Sense vibrations  May be important in “hearing” mates

50. Johnston organ Sound causes segment 3 to rotate relative to segment 2 Responds w/in 1.2 ms Hear with antennae

51. Skin connected to cytoskeleton by receptor MEC - mechanosensory proteins identified from mutants Ion channel Tubulins

52. Cellular structure  Very similar structures to C. elegans mechanoreceptor  No ion channel shown??

53. Possible channel - nompC

54. NOMPC

55. NompC is new member of the TRP family of ion channels

56. Other paper we will read for Wed