Lecture 12 : Metabotropic signaling and mechanoreceptors Fain begin ch 5 10/12/09.
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Transcript of Lecture 12 : Metabotropic signaling and mechanoreceptors Fain begin ch 5 10/12/09.
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Lecture 12 : Metabotropic Lecture 12 : Metabotropic signaling and signaling and
mechanoreceptorsmechanoreceptorsFain begin ch 5
10/12/09
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2009 Nobel prize in 2009 Nobel prize in LiteratureLiterature
Writes about life in Romania under a dictatorship German minority
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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
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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)
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Signal transductionSignal transduction
IonotropicDirectly gate ion channel
MetabotropicGate ion channel through a G protein
and 2nd messenger
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GPCR signalingGPCR signaling
GDP
GPCR*
+
M=2nd messenger sends signal to ion channel
M
Receptor
G protein
Effector
GTP
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MechanoreceptorsFain ch 5
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MechanoreceptionMechanoreception
Responds to mechanical pressure or distortionHearingTouchAcceleration detection
Why are ionotropic mechanisms good for mechanoreception?
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Express and patch clamp piece Express and patch clamp piece of membrane - record current as of membrane - record current as
apply pressureapply pressure
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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
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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
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Anishkin and Sukharev 2009Anishkin and Sukharev 2009
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Gillespie and Walker 2001Gillespie and Walker 2001
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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
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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
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Goals for mechanosensationGoals for mechanosensation
1. Maximize speed of signal detection
2. Maximize sensitivity of response
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Paramecium sense of touchParamecium sense of touch
Differential response:
If touch front, reverses direction, turn and go another way
If touch back, swims faster
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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
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Genomics of Paramecium Genomics of Paramecium sensory receptors???sensory receptors???
72 Mb40,000 genes
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C. elegansC. elegans
The worm959 cells302 neurons
Many methods available for studying pathways
C. briggsae is closely related worm
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Forward genetics approachForward genetics approach
Find or make mutants with particular phenotypeChemical mutagen
ENUN-ethyl N-nitrosourea
Transposons
Normal
Mutant
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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
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Li et al 2006 : Sixth sense in wormsLi et al 2006 : Sixth sense in worms
One of papers we will read for Wed
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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
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C. elegansC. elegans methods are worked methods are worked outout
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C. elegansC. elegans
Touch frontWorm moves backward
Touch backWorm moves forward
Touch middleNo effect
C. briggsae is closely related worm
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WormbookWormbook
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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
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Wormbase describes all 959 Wormbase describes all 959 cells…cells…
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..including its cell lineage
But not what it’s name means!!!
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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
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Skin connected to Skin connected to cytoskeleton by receptorcytoskeleton by receptor
MEC - mechanosensory proteins identified from mutants
Ion channel
Tubulins
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MechanoreceptorMechanoreceptor
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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
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CrayfishCrayfish
Large enough for intracellular recordingGenetics are difficult
Abdominal stretch receptorsMRO = muscle receptor organ
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Stretch receptorStretch receptor
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Crayfish mechanoreceptorCrayfish mechanoreceptor
Intracellular recording from cell attached to muscle
Stretch muscle and record
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Depolarize and generate action potentialsDepolarize and generate action potentials
MRO1 - continuous response during stretch - slow adapt
MRO2 - respond only at first - fast adapt
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Another differences in MROAnother differences in MRO11 and MROand MRO22 adaptation adaptation
Also differences under voltage clamped conditionsRate of fiber relaxationGreater adaptation in MRO2
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Crayfish stretch receptorsCrayfish stretch receptors
May be directly responding to membrane stretch
To test this, pull off patch and apply pressure to see response
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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
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Cray fish genomics?Cray fish genomics?
Genome size 5-6 Gb
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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
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Type I - Hair plate sensillumType I - Hair plate sensillum
Outer segmentConnects to base of bristleMicrotubules surround by extracellular matrix
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Hair cell sensillumHair cell sensillum
Bristle motion causes cuticle to push on cap
Cap pushes on tubular bodyDepolarizationLikely cation channels open
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Type I - Campaniform Type I - Campaniform sensillumsensillum
Senses compression of cuticle
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Type I - Scolopidial organType I - Scolopidial organ
Scolopale cell - supporting cellSecretes extracellullar membrane
Detect vibrations transmitted from cuticle through accessory cellTympanal organsJohnston’s organ
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Johnston’s organJohnston’s organ
Located in antennae
Sense vibrations May be important
in “hearing” mates
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Johnston organ
Sound causes segment 3 to rotate relative to segment 2
Responds w/in 1.2 ms
Hear with antennae
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Skin connected to Skin connected to cytoskeleton by receptorcytoskeleton by receptor
MEC - mechanosensory proteins identified from mutants
Ion channel
Tubulins
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Cellular structureCellular structure
Very similar structures to C. elegans mechanoreceptor No ion channel shown??
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Possible channel - nompCPossible channel - nompC
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NOMPCNOMPC
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NompC is new member of the TRP NompC is new member of the TRP family of ion channelsfamily of ion channels
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Other paper we will read for WedOther paper we will read for Wed