Exam III Flashcards
2 broad categories of senses
- Special senses - HSTV hearing, smell, taste, vision
2. Somatic senses - TTPP touch, temp, pain, proprioception
What is sound?
Vibration of air
What is light
EM waves
Process of signal transduction
- Stimulus comes in form of energy
- Sensory potentials
- APs
- Brain interpretation
Signal transduction pathway
- Stimulus activates sensory Rs
- Sensory Rs act as signal transducers
- These primary sensory neurons project into CNS, and connect to 2° sensory neurons
- Which then project to various cortical regions
If APs are more or less the same, how does our brain perceive a particular stimulus?
- Location of stimulus
- Type of Rs activates
–> tell the brain what the signal is
3 types of stimuli
- Mechanical (touch, hearing, temp, noxious)
- Electromagnetic
- Chemical (smell, taste)
What do sensory Rs do?
Signal transducers
Convert energy stimuli into electrical signals – receptor potentials – and when large enough, trigger AP
Stimulus has 4 attributes that the brain can register
MILT
- Modality (quality) - depends on physical-chemical energy
- Intensity - coded by # of Rs activated
- Location - topography, vision field, hair displacement/act
- Timing - speed + duration
To encode modality…
Stimulus must be
- adequate
- threshold
PhotoRs
4 different photoRs
Activated by light at different wavelengths
Hair cells
Found in cochlea
Have different sensitivities based on location
Encoding of intensity
# of Rs AP frequency
Encoding of location
What part touched, vision field, hair cells activated
Encoding of timing
- Tonic Rs - slowly adapting
2. Phasic Rs - fast-adapting
Where does phototransduction take place?
Retina
Steps of vision
- [PHYSICAL] Light –> eye –> focused on the retina
- [PHOTOTRANSDUCTION]
- Processing of visual info by retina and brain
Eye anatomy
Retina detects light (back of eye)
PhotoRs located on retina
Lens on front focuses light to retina, iris can contract/dilate to let less/more light in
Retina
Back of eye
Has photoRs - primary efferent ganglion cells
2 types of photoreceptors
Rods - low light, no color - on edge
Cones - higher light, color, spatial acuity - in center
Retina has how many types of…
4 types of photoRs
- 1 type of rod, 3 types of cones (respond to diff λ)
Rods and cones can be divided into 3 components
- Outer segment - light sensitive part; many disks that contain photopigment (invaginations of PM in cone, pinched off in rods)
- Inner segment (contains nucleus and cells)
- Synaptic terminals (contain NTs, release GLUT; project to bipolar cells, which express various types of GLUT Rs, can dep./hyp)
Rods and cones have what type of potentials?
Only R potentials, cannot fire AP
Directly release NTs
Rods v. cones
- Many more rods (20:1)
- Rods have more photopigment, more disks
- Rods have higher convergence
(many rods converge onto bipolar cell)
(cones almost always make 1:1:1 connections) - Rods more sensitive to light
- Rods have low acuity
- Rods = no color (mono), cones = color
Rod v. cone current
Rod current responds and decays slowly
Cone = fast response, fast decay
Light produces current via
- Biochemical pathway leads to ↓ in cGMP
2. Closure of cGMP-gated channels
Cone RMP
-40mV
Light hyperpolarizes cell at increasing intensities
Why do cone cells have an RMP so much less negative than other?
RMP normally set by leakage of K+ channels
-40 far away from that
Other channels must be open in the dark
Phototransduction in retina process
Within the membrane of disks…
- there are rhodopsins (they are photopigments – they are GCPRs with a light-sensitive mol covalently attached to it)
- Rhodopsin is coupled to a G protein called Transducin
- Transducin activates enzyme PDE, which breaks down cGMP
- cGMP binds to CNG channels (nonselective cation channels that conduct Na+/Ca into cell on outer PM)
What is the “dark current”?
In the dark, [cGMP] in cytoplasm is HIGH
- cGMP binds to CNG channels, activating/opening them
bc these channels are continuously open, RMP is much less neg (↑ Na+/Ca2+), at -40 mV
In inner segment, there is K+ channel taking K+ out (not sensitive to light) which helps to balance
CNG channels
Present in membrane outer segment
Conduct Na+/Ca into cell
What is the lumen of the disk like?
More/less like extracellular environment
What happens when light is shined?
Rhodopsin –> Transducin –> PDE –> Breakdown/↓ in cGMP –> channels begin to close –> hyperpolarization
….dark current abolished by light
Within disks (outer segment of rods/cones)... Rhodopsins (photopigments, GCPRs with light-sensitive mol covalently attached) --> coupled to a G protein called Transducin --> Transducin activates enzyme PDE, which breaks down cGMP --> cGMP unable to bind to/activate Na+/Ca channels, making the cell more neg (hyper.)
What does cGMP do in phototransduction pathway?
cGMP binds to CNG channels (nonselective cation channels that conduct Na+/Ca into cell)
Located on PM of outer segment in rods/cones within retina
When dark current is abolished, what channels are open/closed?
CNG channels (which conduct Na+/Ca into cell) are closed due to ↓ in cGMP
K+ channels on inner segment is still conducting K+ out, which will reduce MP to a neg level
Rhodopsin
Photopigments – they are GCPRs with a light-sensitive mol covalently attached to it
2 parts:
- opsin (GCPR)
- 11-cis-retinal
cis configuration in dark (inactive)
trans configuration in light (active)
Δ in retinal from cis->trans –> confirmation Δ of opsin –> which allows it to activate transducin
Retinol recycled
What happens if there’s a mutation in the CNG channel?
W/o channels, there’s no phototransduction
Responsible for light –> electrical signal
NT-release in phototransduction dependent on Ca2+, which CNG channels bring into the cell
NT-release in phototransduction
Dependent on Ca2+
NTs continuously released in the dark
Different Rs will interpret the NT as hyper./dep.
NT type????? GLUT?
Amplification in phototransduction
OP
Bc light activates a biochemical cascade, there’s a lot of amp
*OPSIN can activate 800 g-proteins
g-protein activates PDE 1:1
*PDE can hydrolyze 6 GMP molecules
Absorption of 1 photon ~200 CNG channels
What is mainly responsible for dark current?
CNG channels mainly conduct Na+
(mainly responsible for dark current)
A little bit of Ca gets in too
What does Ca2+ do in phototransduction
Slightly contributes to dark current through CNG channels (although mostly Na does this)
In dark, a little Ca gets into cell through CNG
- Ca reduces sensitivity of CNG channels to cGMP
….since cGMP opens CNG channels…now less Na/Ca+ is getting into cell - Ca inhibits the enzyme guanylatecyclase (which turns GTP -> GMP)
….less Na/Ca+, less dark current - Ca inhibits rhodopsin kinase (which P rhodopsin)
…inactivates light cascade
Turn on light…
Light will close CNG channels, meaning no Ca/Na influx
- ↑ in cGMP (bc Ca not blocking guanylatecyclase (GTP -> GMP) - ↑ in CNG sensitivity to cGMP => REDUCED SENSITIVITY TO LIGHT! due to ↑ cGMP, ↓ rhodopsin activity
Allows cells to react increasingly to light
How do we see color?
We see color bc we have 3 different types of cones
which are activated by 3 different wavelengths of light
- each cone has a different tuning curve for light
How do cones respond to various wavelengths of light?
B, G, and R cones…
Have different types of opsins
Retinol same, opsins different
(Different opsin AA sequences)
Obsins AA comparison
Red and green obsins = highly similar
just a handful of different AAs
Role of AAs in photopigments
Make photopigments respond to green v. red v. blue light
Distribution of red, blue and green cones in retina
Mostly red and green
Only 5-10% blue
Red/green ratio vary, but does not affect color perception
Causes of color blindness
- Lacking 1+ types of cones
-> caused either by
degeneration of cones
CNG channel mutation - Mutation in cone photopigments
shifts tuning curve –> partial/total colorblindess - Damage to visual cortex
Mutations of CNG channels
Causes partial or total color blindness
NT release reduction in vision
Reduction of dark current and continuous efflux causes mem. hyperpolarization
—> Reducing GLUT release
What reversal potential does Na+ give you
+60 mv
What reversal potential does Ca2+ give you
+60 mv
Which molecules can give you rev potential of +60 mv?
Na+ and Ca2+
What is hearing?
Perception of sound energy
What is sound?
Compression/decompression of air
What does a tuning fork do?
Causes vibration of air
3 properties of sound
- Pitch/tone: determined by frequency*
- Intensity/loudness: measured in dB
- Timbre/quality: based on overtones
- frequency: how many waves per 1 second (Hz)
ex: 5 waves per 1 sec = 5 Hz
What is frequency (in regard to sound)?
- frequency: how many waves per 1 secondunit - Hz
ex: 5 waves per 1 sec = 5 Hz
What happens to our hearing ability as we age?
We lose ability to hear very low and very high frequencies
How are dB measured?
Log form
We can perceive a very large range of sound intensity/loudness, so we put it in log
Normal convo: 60 db (1m x higher than threshold)
Rock concert: 120 db (1 tril x higher than threshold)
What do very high sounds do?
Damage hair cells responsible for transduction
What does outer ear do?
Outer: sound conduction
Sound vibration traveling - sound transduction
Ear vibration –> tympanic mem vibration –> 3 bones vibration –> pushes on oval window –> vibration transmits waves of sound into the cochlear fluid –> fluid vibration causes movement of tectoral+basilar membrane –> bending of hair cells causes transduction
Ear anatomy
Outer ear –> tympanic membrane –> 3 bones of middle ear –> oval window –> cochlea
Sound mechotransduction occurs in the cochlea
Steps in sound mechanotransduction
(1) air wave → (2) mechanical vibration of the ear bones → (3) fluid vibration in the cochlea→ (4) movement between tectorial and Basilar membranes → (5) RP in IHCs → (6) release of NT from IHCs→ (7) AP firing in the auditory nerve
Cochlea structure
Has 3 fluid-filled compartments
- Vestibular duct
- Cochlear duct
- Tympanic duct
Cochlear duct
Middle fluid filled
Contains endolymph (fluid that is similar to cyto, high in K+)
~140mm [K+]
Vestibular duct
Top fluid filled
Contains perilymph (fluid that is similar to extracell/IF, high in Na+)
140 mm Na - High
2 mm Ca - Normal
10 mm K - Low
Organ of Corti
Sensitive element in the inner ear and can be thought of as the body’s microphone
In b/w tectoral and basilar membrane
4 rows of hair cells protrude
3 rows OHC, 1 row IHC
OHC: finetune responses of IHC, sharpen frequency
IHC: signal transduction/turn vibration into sound
these hair cells' bodies are anchored on basilar mem
IHCs make synaptic connections with auditory nerves and release NTs that go to brain
OHCs innervated by efferent nerves (outward innervation)
Inner hair cell bundles
Consists of 2 types of cilia
- kinocilia (only 1 per bundle)
- stereocilia (20-50 per bundle)
Cilia embedded in tectoral membrane, particularly the taller ones
Kinos tall, stereos shorter
Hair cells on bm
Close to oval window: respond to high frequencies
Close to cochlear apex/end: respond to low freq
Endocochlear potential
In IHCs…
Hair bundles immersed in endolymph
Basolateral side immersed in perilymph
Large voltage difference
+ 80 mv in endo
0 in peri (ref point)
-50 in IHC cyto
Meaning endolymph much more positive
This is the endocochlear potential
- Inside of IHC is negative
large voltage difference b/w endolymph and IHC cyto
Tight junctions prevent exchange of ions b/w
endo/IHC extracell./paralymph (sealed up)
in b/w hair cells and other cells
IHCs release NTs - cause R potentials
glutamate binds to ionotropic Rs in afferent nerve endings
How does bending of IHCs produce R potential?
Bundles the stereocilia are often lined up in rows of increasing height, similar to a staircase
Tiny springs, called tip links, connect the tips of stereocilia
run upward from shortest to tallest
streched when pushed from short->long
When stretched (pos), they open ion channels that are located on tip of stereocilia - these nonselective cation channels conduct K+
Even though [K+] is same in endolymph and IHC cyto, due to very large voltage difference (+80 in endo, -50 mV in IHC cyto) =====> this creates a very large driving force
--- > K+ pushed in endo->cyto causes depolarization
When hair bundles pushed other way (neg), channels close, causes hyperpolarization (some ~15% channels open at rest)
Voltage/[ ] differences surrounding IHCs
Endolymph (fluid that is similar to cyto, high in K+)
~140mm [K+]
+80 mV
Perilymph (fluid that is similar to extracell/IF, high in Na+)
140 mm Na - High
2 mm Ca - Normal
10 mm K - Low
0 mV
IHC cyto
~140/5mm [K+]
-50 mV
Large voltage difference
+ 80 mv in endo
0 in peri (ref point)
-50 in IHC cyto
IHC bending response
- Highly sensitive
- 3 nm (diam of K+ ion) enough stretch to open/close channels
- Extremely fast
direct, can open in 10 μs
Tip links - function and structure
Tiny springs that connect tips of stereocilia
Run upward from shortest to tallest (L->R)
streched when pushed from short->long
Tip links made of protein
Ca+ important for integrity
Ca+ chelators destroy tip links and destroy sound transduction
Each tip link has ~100 ion channels
Composed of at least 2 proteins
bundle together to form dimer, dimers bundle together to form tip link
Cadherin21 and Protocadherin15
What are tip links composed of?
Cadherin21 and Protocadherin15
What are the channels in tip links?
TCM1 is the mechanotransduction channel
found in IHCs
has 10 transmem segments
believed to be a dimer with pore
nonselective cation channel, conducts K+ into IHC
TCM1
TCM1 is the mechanotransduction channel
found in IHCs
has 10 transmem segments
believed to be a dimer with pore
nonselective cation channel, conducts K+ into IHC
What NT do IHCs release?
Glutamate
IHC at rest
Some (about 15%) channels open at rest
- -> Constant leakage of Ca2+ into IHCs
- -> Constitutive release of NTS
Spontaneous firing of the auditory nerve
Explains why relaxation causes hyperpolarization (the little bit of K that was getting in, making IHC less neg, is now blocked)
IHCs can respond…
Bidirectionally
Different responses to bending
- Short->tall : stretch tip links -> open channels -> dep.
- Tall->short : relax tip links -> close channels -> hyp
(kino-stereo push KINOS tall, stereos short) - 90° push : don’t really cause bend, very little effect
Hair bundle displacement v. R potential
Graded responses
Larger response w/more push
How to encode for frequency?
Related to physical properties of basilar membrane
Flatten out –> base/oval: thick + rigid
apex/end: thin + flexible
~30 mm long in total
Stiff/oval window: respond to high frequencies
Flimsy/apex: respond to low freq
Human hearing range
20 Hz - 20,000 Hz
How is frequency encoded in hair cells?
Location on basilar membrane
- Hair cells on different points on basilar mem have different compositions of ion channels
Each has different intrinsic oscillation freq - characteristic freq
If sound wave matches that particular hair cell’s freq, produces larger response
Sound waves at the characteristic frequency of a cell cause the largest fluctuation in membrane potential.
DUE TO DIFFERENT SETS OF CHANNELS
How is intensity encoded in hair cells?
Louder sound = larger R potential = more NT release = faster firing of APs (in auditory nerve)
What do the 3 rows of OHC in the organ of Corti do?
Not sensory neurons themselves, but do play important role
Through contractions/extensions, can alter stiffness of TECTORAL membrane
.˙. finetune
contract to sound stimulation + efferent innervation
respond to voltage Δs with contraction
“Dancing” hair cell
Add electrode + whole-cell voltage clamp –> pass current into hair cell –> Δ in voltage –> hair cell contracts
Causes of deafness
CSC
- Conductive deafness - has to do w/sound conductance itself
- Sensorineural deafness - problem with sound transduction pathway
- issue in organ of Corti, hair cell degeneration
- particularly at high freq., we only have 30,000 IHCs die with age (rock concert -> constant stretching -> break more easily) - Or too much Ca2+ -> causes cell damage
Which do we have more of - OHCs or IHCs?
Much more OHCs
~4x more
Distribution of hair cells along BM
Pretty much uniform
Resolving power
Resolving power much lower at high frequency
Ex: Can’t distinguish b/w 19,000 to 20,000 Hz, but can differentiate 200 to 210 Hz
Chemosensation
Smell + taste
Primitive sense, exists even in single-celled organisms (ex: bacteria)
Where does transduction take place for smell?
Olfactory epithelium
Olfactory R neurons (ORNS
Bipolar sensory neurons containing olfactory Rs
Concentrated in cilia
Can fire APs
Die - must be constantly regenerated (same w/taste cells)
Apical side: numerous cilia embedded in mucus
Basolateral: project w/long axons to olfactory bulb
ORs are GCPRs
ORN regeneration
Stem cells produce basal cells -> basal cells produce ORNs
Regenerate and express same # and types
ORN number comparison
More ORNs = better smeller
Humans = 12 m Rat = 15 m (olfactory bulb much larger comp.) Dog = 1 b
Cell types in olfactory epithelium and their functions
Olfactory receptor neurons (ORNs)
- Bipolar sensory neurons containing olfactory Rs
- Can fire APs
Basal cells
- dividing stem cells
- generate new ORNs
Supporting cells
- detoxify dangerous chemicals
Bowman’s gland
- secretes mucus
Human odorant molecule detection threshold
0.1 nm –> we can detect at very low [ ]
What is important for odorant detection?
Cilia
- odorant molecules dissolved in saliva
- bind to Rs in olfactory cilia
Test: How do we know cilia of ORNs respond to odorants?
- Isolate ORNs
- Record currents elicited by various chemicals
whole-cell voltage clamp
inward current produces depolarization
In cilia: we see large inward current
In soma: we see little reaction
[Shows that ORN are concentrated in cilia]
Olfactory R genes
Buck and Axel - First to clone OR genes
Found OR genes are GCPRs
These genes form large gene family, largest gene family in many species
3-5% of all genes
Mammalian olfactory R genes /= introns
No alternative splicing, all linked together
Gene is transcribed in full, in one piece in mammals
Many of them are pseudogenes
Expression is mono-allelic either express mother or father
AC3
Adenyl cyclase 3
Cilia marker
Found only in apical epithelia, in cilia
A key enzyme mediating the cAMP signaling in neuronal cilia
Turns ATP -> cAMP
Zoning v. non-zoning distribution
Lect. 18 [12/23]
Olfactory transduction
- Odorant brings to R (GCPR)
- Golf (now GTP-bound) activates AC3
- AC3 [ATP -> cAMP]
- cAMP activates nonselective cation channel
conduct Na+ and Ca in
produces inward current
—> depolarization —> R potential
if large enough, R potential will produce AP - Ca can flow through + activate Cl-gated channels
Cl- will then flow out, helping to depolarize more
Depolarization = transduction of smell
of human odorant Rs
~1,000 different odorant protein Rs
Why attempts to express olfactory receptors in heterologous systems have failed?
In order to produce function, you need the pathway, not just the R alone. Need Gprotein (Golf), AC3, the channel…
Expressing R alone will not be enough
What happens if you knock out components of olfactory transduction pathway?
Abolish olfactory signal transduction
How are olfactory transduction responses turned off?
↓ [ ] of odorants, or move away from source
Binding will stop, process will end
How does smell desensitization work?
- ↓ [cAMP] through hydrolysis by PDE
then cation channel inactivated - Ca can bind to calmodulin, which can bind to both channels (Na/Ca+, Cl-) to close them
- Over time, [Ca} ↓ through a Na/Ca exchanger, leading to reduced inward current, eventually ending process
ORN responses
Some are specific, some are general
APs are concentration-dependent, more [ ] = faster firing
spontaneous alone
[19/23] Lect. 18
Where do ORNs connect to?
ORNS send axons to the olfactory bulb
- axons make connections with 2 types of cells in olf. bulb
(1) mitral cells*
(2) tufted cells
this occurs within the glomerulus, within the olf. bulb
*mitral cells - main projection neurons of the olf. bulb
Pair + project
- ORNs expressed in the same R (localized in same area) pair + project to glomerulus together
Glomerulus
The glomerulus is located within the olfactory bulb of the brain where synapses form between the terminals of the olfactory nerve and the dendrites of mitral, periglomerular and tufted cells.
What is the idea of “pair and project”?
Pair + project
- ORNs expressed in the same R (localized in same area) pair + project to glomerulus together
Anosmia
Loss of sense of smell
- can be caused by brain damage, virus, or age, or congenital
Due to abnormal development of olf system
Odorant molecules vs. taste molecules
Odorants are often volatile airborne molecules
Tastants are often nonvolatile, soluble molecules
NT in each type, and dep. v hyp.
x
Human sensitivity to tastants
Quite weak - we need high [ ] to taste
The threshold concentration for tastantsis usually high
But for bitter substances, threshold is low
5 taste modalities
Sour Salty Bitter Sweet Umami (glutamate)
5 taste modalities associated w/essential body functions
Sour/salty - essential ions
Bitter - warning of toxicity
Sweet/umami - food intake
Taste buds
Found in papilla
Different papilla have different buds
*add more
Three cell types in taste buds
- Taste cells
- Supporting cells
- Basal cells (give rise to mature taste cells)
Lifespan of taste cells
~1 month
Taste cells
Taste receptor cells are
(1) polarized epithelial cells
have apical/bl sides, .˙. polarized
(2) short lived (~1 month)
(3) can be regenerated from basal cells
(4) have voltage-gated Na+, K+, and Ca2+ channels, release on bl side, and thus can fire action potentials
NT believed to be serotonin
“Tongue map” argument
Five taste modalities are diffusely distributed on the tongue
There is no “tongue map”
How do taste cells perceive different tastes?
3 encoding theories
(1) Labeled-line model
- everything predetermined
- each cell tuned to respond to certain taste
- each cell connected to certain gustatory nerve
(2) Cross-fibre model A
- taste cells can respond to all 5 tastes
- connected to certain gustatory nerve
- coding occurs in CNS
(3) Cross-fibre model B
- taste cells can respond to all 5 tastes
- connected to all gustatory nerves
- coding occurs in CNS
Labeled line = correct
Taste transduction
Tastants depolarize via 2 mechanisms
(1) direct permeation
(2) indirect activation of GCPRs
NT believed to be serotonin
- Trigger zone is not axon hillock (far away), it’s very close to nerve terminals, when terminals sufficiently depolarized, produce AP
Taste transduction via ion channels
Salty channel conducts salt
Sour channel conducts protons
- channel formed by PKD1L3 and TrpP3
*more protons
What is sour taste channel formed by?
PKD1L3 and TrpP3
OTOP1
Proton channel important for sour taste
- Whole-cell recording from isolated taste cells
Find that low pH can produce large current
at neutral pH - normal current
at low pH - large inward current - Knock channels out, do whole-cell recording from isolated taste cells again
Add low pH - no response
.˙. we know these channels needed
Evidence good for OTOP1 being proton channel
Taste transduction via GPCRs
Only 3 T1Rs. Why such a small number?
Knock-out of T1R2 attenuates sweet taste; umamitaste remains unaffected.
Knock-out of T1R1 abolishes umamitaste but leaves sweet taste intact.
Knock-out of T1R3 attenuates both sweet and umami taste
TRPM5 channels are temperature sensitive –is this related to temperature sensitivity of certain types of taste sensation?
Taste Rs for 5 senses
Salty - ion channel
Sour - ion channel (PKD1L3 and TRPP3)
Bitter - GCPR (monomer T2R)
Sweet - GCPR (dimer T1R2 and T1R3)
Umami - GCPR (dimer T1R1 and T1R3)
Types of taste receptor proteins
There are 4 in total
- T1R
- T1R1, T1R2, T1R3
- T2R
Taste transduction pathway for GCPRs
GCPR activated -> coupled to g-protein Gustducin -> Bind to PLCβ2 (breaks down lipids [PIP2] to produce two 2° msngrs, dieserglycerol and IP3
IP3 then binds to TRMP5 channel, which causes influx of Ca
Main evidence for taste pathway components
Knock out experiments
Knock out experiments for sweet/umami taste
Knock-out of T1R2 abolishes sweet taste; umami taste unaffected
Knock-out of T1R1 abolishes umami taste; sweet taste unaffected
Knock-out of T1R3 abolishes both sweet and umami taste
TRPM5
Temp. sensitive cation channel., conducts Ca2+ influx
What do cats not respond to?
Cats don’t have sweet Rs
T1R2 gene mutated (pseudogene)
Sweet taste R
Sweet - GCPR (dimer T1R2 and T1R3)
Umami taste R
Umami - GCPR (dimer T1R1 and T1R3)
Bitter taste R
Bitter taste cells express ~30 T2Rs
Why so many?
- you want more Rs to detect more varieties of potentially harmful substances
We think T2Rs work as monomers
Which tastes use GCPRs
Bitter, sweet, umami
Only difference is T R type
Taste R localization
Different taste receptor proteins are expressed in different taste receptor cells
Panda taste
umami not present
Sea animals
Sweet and umami = pseudogenes
Bc they swallow their food whole
Knock outs of various components
Slide 22/30 Lect. 19
Support labeled model line
Support for labeled model line
Slide 22/30 Lect. 19
(1) Different taste receptor proteins are expressed in different taste receptor cells
(2) Knock out of a particular taste receptor protein results in the deficit of a specific taste modality
(3) Specific ablation of T1R2-, T2R-and PKD1L3-expressing cells results in the loss of a single taste quality (sweet, bitter and sour, respectively).(Genetically targeting diphtheria toxin to a defined subset of taste receptor cells)
(4) Knock out of PLCB2or TRPM5 eliminates sweet, bitter and umami taste, but not salty and sour taste.(5)Selectively expressing PLCB2 in T2R-expressing neurons in PLCB2 knock-out mice restores bitter taste, but not sweet and umami taste.
(6) Selectively expressing a taste-unrelated membrane receptor, which responds to a synthetic tasteless ligand, in sweet TRCs results in attraction of the engineered mice to the ligand. Conversely, expressing this receptor in bitter TRCs results in avoidance
(7) Expressing a novel bitter receptor in sweet cells results in attraction of the transgenic mice to a bitter compound (see figure in next slide), indicating that taste quality is determined by the TRC type, not by the taste receptor proteins or even the tastant molecules.
TRPM5, PLCβ2 experiment in T2R cells
Rats drinking
Dissolve various tastes in H20
- w/higher [ ], they drink more/less
- Knock out TRPM5
==> TRPM5 is the taste transduction channel for sweet, umami and bitter taste
When knocked out, rats don’t show [ ] dependence to bitter/sweet/umami
- Knock out PLCβ2
==> PLCβ2 is an essential transduction molecule for sweet, umami and bitter taste
No [ ] dependence (to bitter/sweet/umami)
Put it back, response returns
Expressing PLCβ2 in T2R taste receptors cells restores bitter taste but not sweet or umami taste, supporting the notion that individual R cells are tuned to a single taste quality
Engineering bitter R
Developed novel (recombinant) bitter R
responsive to particular molecule
- Feed molecule to wild-type animal
- no [ ] difference/response, bc don’t have R
- Then express R on animal’s bitter cells, feed molecule
- animals avoid compound
- Now express R on animal’s sweet cells, feed molecule
- animals love it
As long as you activate that R, you get the taste of the cell
- animals love it
Supports labeled-line model
Miracle fruit
Makes sour -> sweet
Active ingredient : protein called miraculin (turns sour->sweet)
Miraculin itself does not taste sweet
- When taste buds are exposed to miraculin, the protein binds to the sweetness receptors
This causes normally-sour-tasting acidic foods, such as citrus, to be perceived as sweet
How does miraculin change sour to sweet?
At low pH, miraculin Δs its confirmation to bind to sweet R
Binds to T1R2, where aspartame binds (art. sweetener)
=> causing sweet taste
Reduces effectiveness of sweeteners
At neutral pH, doesn’t do anything
=> Antagonist at neutral pH and functionally changes into an agonist at acidic pH
Somatosensation
Touch, thermosensation, pain
Somatosensory Rs
Touch, thermosensation, pain
Project to DRG nuerons
Touch R types
Mediated by 4 Rs
Myelinated, large axons
==> fast conduction velocity
Axon comparison
x
Which senses the fastest?
Pain Rs
These Rs are basically free nerve endings (pretty much on skin surface)
Sharp pain = Aδ axons (myelinated, thinnish)
Slow/chronic pain = C axons (unmyelinated, thinnest)
How do touch Rs respond to mechanical stimuli?
By expressing channels that are directly activated by membrane stretch
Ion channels are directly on PM
==> Transduction of mechanical force –> electrical signal
Mediated by mechanosensitive ion channels
Membrane stretch –> Channels directly opened –> Na influx –> Produce R potentials, depolarization –> AP firing (close to nerve ending)
(Nonselective cation channels), (ion channels mostly in nerve endings)
Piezo channels
Piezo channel types respond to various membrane stretch
Inward current produced
- amp of current depends on mag. of pressure
Current profile:
- respond to stronger mechanosensation
- respond quickly (fast rise/decay)
Piezo channel structure
Trimer w hole in middle
Channel can bend membrane
How do we know Piezo channels are mechanosensitive
Purify protein -> place Piezo into lipid bilayer (no other components present) -> bend lipid bilayer (bending = mechanical force) -> record channel current
Piezo channels directly respond to mech. force
When Peizo2 knocked out of DRG neurons, no current
Piezo mouse experiment
- Wild type - Piezo2 induces current
- Piezo2 KO in DRG neurons - no current
(in some DRG neurons, Piezo needed)
(touch sensitivity goes down, Piezo partly responsible for touch sensation) - Piezo2 KO, overexpress Piezo1 (will it rescue current?)
(Yes!) - Express both Piezo1 + Piezo 2
(Increased sensitivity to touch)
==> Overall, both important for touch, both mechanosensitive
Conduct inward current -> depolar. -> AP
DRG axons for temp
Aδ axons (myelinated, thinnish)
C axons (unmyelinated, thinnest)
2 temperature Rs
TrpV1
- capsaicin R, WARM/HOT temp
TrpM8
- cold temp
==> These channels express ion channels that are sensitive to different temps
=> Differential expression of Na+and K+channels modulates the temperature thresholds
Rs/channels located at the free nerve endings of DRG neurons)
Minty gum perception
TrpM8 underlies cool feeling w/gum
TrpV1 activated
Can bind to capsaicin to be activated
Can be activated by heat
Can be modulated by protons (larger response at lower pH)
Spiciness
Not a “taste”
Mediated by pain Rs responsive to the face
Rs not found in taste buds or gustatory nerve endings
Capsaicin molecular structure
Hydrophobic
Cold water somewhat helps, doesn’t remove it very effectively
MILK = best –> more fat, fat better dissolves hydrophobic substances
Mice v. tree shrews
If you feed spicy mol (Cap2) to mice, with higher [ ] eat less
If you feed spicy mol (Cap2) to tree shrews, with higher [ ] eat more until reach high threshold (then slightly decline)
==> Then compare TrpV1 channels
transfect into kidney cells, record channels
Cap2 activates mice V1 Rs at low [ ]
Tree shrews need much higher [ ] before rejecting
Why?
- Single AA mutation in cap-binding pocket that decreases sensitivity
If you put tree shrew AAs in mouse, you dramatically ↓ sensitivity
TrpV1 is activated by…
high temperature
Hardly no current under 35/35 C
Strongly activated by rising temps
Camel and squirrel TrpV1 response profiles
Hot temp response
Camel and squirrel don’t respond to high temps, have high threshold/tolerance
Due to AA difference
How do we know TrPV1 is an inherently heat-sensitive channel?
Purify protein -> place TrPV1 it in lipid bilayer (no other proteins present) -> Δ temp -> record channel current
At low temp, little/no response
At higher temps, increasingly large response
Channel protein itself activated by rising temp
(assumed to be due to AA sequence)
TrpM8 activated by….
Activated by menthol and other cooling agents
Icilin = largest response, much more effective in activating M8 than menthol
No current at body temp
Current gets bigger+bigger with lower temps
Pain-sensing DRG neuron groups
MTP
- Mechanical - high mech. force
- Thermal - above 35 C, below 5 C
- Polymodal - high mech. force, extreme temp (high freq.)
All DRG?
x
Channels most widely expressed in nociceptive neurons
TrpV1
TrpA1
ACIC
All nonselective cation channels
Produce R potential
NaV 1.7
Na channel that seems to be vital for pain reception
Mutation of channel -> no pain of any type
These patients basically don’t express NaV 1.7 channels
w/o NaV 1.7, no APs
PKA phosphorylation can modulate response, but cannot alone open channel
Ion channels involved in pain/pathway
TrpV1, TrpA1, ACIC
–> produce R potentials upon noxious stimuli
R potential travels down axon, reaches NaV 1.7
–> produces AP
NTs release to spinal cord
What can modulate NaV 1.7 response
PKA phosphorylation can modulate response, but cannot alone open channel
