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

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

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

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)

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

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

Mechanoreceptors Fain ch 5

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

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

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

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

Anishkin and Sukharev 2009

Gillespie and Walker 2001

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

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

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

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

Paramecium sense of touch  Touch front (anterior) Depolarization Inflow of Ca +2 If > 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

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

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

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

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

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

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

C. elegans methods are worked out

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

Wormbook

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

Wormbase describes all 959 cells…

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

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

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

Mechanoreceptor

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

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

Stretch receptor

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

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

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

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

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

Cray fish genomics?  Genome size 5-6 Gb

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

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

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

Type I - Campaniform sensillum Senses compression of cuticle

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

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

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

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

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

Possible channel - nompC

NOMPC

NompC is new member of the TRP family of ion channels

Other paper we will read for Wed