Lecture 12 : Metabotropic Lecture 12 : Metabotropic signaling and signaling and

mechanoreceptorsmechanoreceptorsFain begin ch 5

10/12/09

2009 Nobel prize in 2009 Nobel prize in LiteratureLiterature

Writes about life in Romania under a dictatorship German minority

Nobel Peace prizeNobel Peace prize

Awarded the prize for who he is and what he hopes to accomplish

Change in attitude and galvanizing the world

Nobel homeworkNobel 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 transductionSignal transduction

IonotropicDirectly gate ion channel

MetabotropicGate ion channel through a G protein

and 2nd messenger

GPCR signalingGPCR signaling

GDP

GPCR*

+

M=2nd messenger sends signal to ion channel

M

Receptor

G protein

Effector

GTP

MechanoreceptorsFain ch 5

MechanoreceptionMechanoreception

Responds to mechanical pressure or distortionHearingTouchAcceleration detection

Why are ionotropic mechanisms good for mechanoreception?

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

apply pressureapply pressure

MscL - Mechanosensative MscL - Mechanosensative channel, largechannel, large

Large conductance

5 subunits x 2 TMsM1 faces poreM2 faces membrane

Hydrophobic pore keeps water from flowing across membrane

Pore opens like iris M1/M2 rotate-Pore opens like iris M1/M2 rotate-Opens S1 helices Opens S1 helices

Sukarev and Ankirin 2004

Channel responds to membrane tensionsOpening enables ion flow/water to relieve osmotic pressure

Anishkin and Sukharev 2009Anishkin and Sukharev 2009

Gillespie and Walker 2001Gillespie and Walker 2001

MechanoreceptionMechanoreception Evolved multiple

timesRecruited different ion

channels each time

3 means of tethering and pulling to open channel• Direct• Indirect through molecule• Indirect through

mechanosensitive protein

QuestionsQuestions

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 mechanosensationGoals for mechanosensation

1. Maximize speed of signal detection

2. Maximize sensitivity of response

Paramecium sense of touchParamecium sense of touch

Differential response:

If touch front, reverses direction, turn and go another way

If touch back, swims faster

Paramecium sense of touchParamecium sense of touch

Touch front (anterior)Depolarization Inflow of Ca+2 If > 10-6 M causes cilia to reverse direction

Touch backHyperpolarizationOutflow of K+

Cilia beat faster Touch middle

Nothing happens

Difficult to figure out what the genes are

Eckert 1972

Genomics of Paramecium Genomics of Paramecium sensory receptors???sensory receptors???

72 Mb40,000 genes

C. elegansC. elegans

The worm959 cells302 neurons

Many methods available for studying pathways

C. briggsae is closely related worm

Forward genetics approachForward genetics approach

Find or make mutants with particular phenotypeChemical mutagen

ENUN-ethyl N-nitrosourea

Transposons

Normal

Mutant

Forward genetics approachForward 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 wormsLi et al 2006 : Sixth sense in worms

One of papers we will read for Wed

Reverse geneticsReverse genetics

Have the genes and need to figure out what they do

Make GFP reportersSee where gene is expressed

Make knock-outsSee what happens if gene is removed

C. elegansC. elegans methods are worked methods are worked outout

C. elegansC. elegans

Touch frontWorm moves backward

Touch backWorm moves forward

Touch middleNo effect

C. briggsae is closely related worm

WormbookWormbook

C. elegansC. elegans sense of touch sense of touch

6 mechanosensory neuronsAnterior - AVM, ALML, ALMRPosterior - PLML, PLMR

Can ablate cells and see if sense is affected

Front

Wormbase describes all 959 Wormbase describes all 959 cells…cells…

..including its cell lineage

But not what it’s name means!!!

C. elegansC. elegans sense of touch 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 Skin connected to cytoskeleton by receptorcytoskeleton by receptor

MEC - mechanosensory proteins identified from mutants

Ion channel

Tubulins

MechanoreceptorMechanoreceptor

Worm touchWorm touch

Touch causes ion channel to open and cell to depolarize

Uses many proteins which are all necessaryCan make knockins or outs of each gene and

figure out how mechanoreceptor worksBut hard to record from neurons

Ion channel is similar to epithelium Na channelHumans have a dozen of these - likely important

in mechanosensation

CrayfishCrayfish

Large enough for intracellular recordingGenetics are difficult

Abdominal stretch receptorsMRO = muscle receptor organ

Stretch receptorStretch receptor

Crayfish mechanoreceptorCrayfish mechanoreceptor

Intracellular recording from cell attached to muscle

Stretch muscle and record

Depolarize and generate action potentialsDepolarize and generate action potentials

MRO1 - continuous response during stretch - slow adapt

MRO2 - respond only at first - fast adapt

Another differences in MROAnother differences in MRO11 and MROand MRO22 adaptation adaptation

Also differences under voltage clamped conditionsRate of fiber relaxationGreater adaptation in MRO2

Crayfish stretch receptorsCrayfish 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 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?Cray fish genomics?

Genome size 5-6 Gb

Insect mechanoreceptorsInsect mechanoreceptors

Type I BipolarCilium at base of outer segmentExtracellular structuresBathed in high K+ medium secreted by

supporting cells Type II

Multipolar - many dendritesAssociate with internal organs or skinNo supporting cells

Type I - Hair plate sensillumType I - Hair plate sensillum

Outer segmentConnects to base of bristleMicrotubules surround by extracellular matrix

Hair cell sensillumHair cell sensillum

Bristle motion causes cuticle to push on cap

Cap pushes on tubular bodyDepolarizationLikely cation channels open

Type I - Campaniform Type I - Campaniform sensillumsensillum

Senses compression of cuticle

Type I - Scolopidial organType I - Scolopidial organ

Scolopale cell - supporting cellSecretes extracellullar membrane

Detect vibrations transmitted from cuticle through accessory cellTympanal organsJohnston’s organ

Johnston’s organJohnston’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 Skin connected to cytoskeleton by receptorcytoskeleton by receptor

MEC - mechanosensory proteins identified from mutants

Ion channel

Tubulins

Cellular structureCellular structure

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

Possible channel - nompCPossible channel - nompC

NOMPCNOMPC

NompC is new member of the TRP NompC is new member of the TRP family of ion channelsfamily of ion channels

Other paper we will read for WedOther paper we will read for Wed