G protein coupled receptors 3

Question 1

What are sensory GPCRs are responsible for

Answer 1

1. Olfactory perception (smell; odorants )
2. Gustatory perception (taste; tastants )
3. Visual perception (light)
4. Main second messengers are cyclic nucleotides (cAMP, cGMP).

Question 2

What does the olfactory epithelium include

Answer 2

1. olfactory neurons
2. supporting cells
3. basal (stem) cells

Question 3

Where are olfactory receptors found and what is their role

Answer 3

1. Olfactory receptors are found on the modified cilia of olfactory neurons.
2. Increases surface area to capture odorant molecules
3. They are the site of olfactory receptor presentation on the nasal epithelium.

Question 4

How are odorant molecules detected

Answer 4

1. An odorant molecule interacts with a g-protein coupled receptor and this causes exchange of GDP for GTP and Alpha subunit is activated
2. Gs type g protein coupes with adenylyl cyclase which causes production of cyclic AMP from ATP
3. Cyclic amp binds to sodium channels on the plasma membrane of neurons which causes channels to open and sodium to enter the cell
4. Membrane depolarisation and action potential
5. Sends signal to olfactory bulb in brain

Question 5

What are odorants

Answer 5

1. Volatile small molecules

Question 6

Describe the distribution of olfactory receptor genes

Answer 6

1. OR genes found on almost all human chromosomes
2. Current numbers more accurate, based on complete genome sequence
3. Around 400 functional genes and almost 500 non-functional pseudogenes

Question 7

What is the basics of genetic control over OR gene expression

Answer 7

1. Each mature olfactory neuron expresses only one receptor gene.
2. One of the two alleles is randomly silenced.
3. Each cluster is controlled by a single enhancer element which interacts randomly with one receptor gene of the cluster.

Question 8

Describe the refined model taking account of olfactory neuron development from an undifferentiated stem cell

Answer 8

1. In stem cells the OR genes in each cluster are wrapped up in histone proteins with modifications typical of constitutive heterochromatin (H3K9me3 and H4K20me3) except in the region of the enhancer.
2. In the immature neuron the enhancer engages with the promoter of one OR gene in the cluster involving the histone demethylase LSD1 (and likely other proteins).
3. This selection process is relatively slow.
4. Transcription of one OR gene begins through recruitment of both O/E and homeodomain (HD) family transcription factors to the ‘open’ promoter region and this transcription leads to rapid negative feedback that reduces LSD1 levels and activity (limiting the ability for additional OR gene expression to commence.

Question 9

What is difference between immature and mature olfactory neurons

Answer 9

1. Immature neurons can express up to several OR genes on different chromosomes, but all but one is silenced as the cells mature.
2. At early stages in olfactory neuron development one neuron can express several OR genes from different chromosomes.
3. The number falls in late immature neurons to 2-3 and becomes 1 per neuron in mature cells.
4. Note also, multiple OR genes are expressed at a relatively low level in immature neurons compared to the high level expression of the single OR gene in mature neurons.
5. This indicates a further (as yet unknown mechanism) to refine expression from multiple loci to just one during OR neuron development

Question 10

Which taste molecules are sensed by GPCRs

Answer 10

1. Three of the five main classes of taste molecules are sensed by GPCRs: sweet, bitter and umami (MSG).
2. Probably also fatty acids (a sixth class of taste molecule).
3. Sweet, umami and fatty acids are perceived as pleasant at low/moderate, but aversive at high, concentrations.

Question 11

How are taste molecules sensed

Answer 11

1. Many GPCRs couple to a few Ga gustducin G proteins.

2. The papilla are what give the tongue its bobbly surface texture, each covered in hundreds of taste buds.

Question 12

Describe experiment that shows Ga gustducin is important for discrimination of bitter and sweet tastes

Answer 12

1. Despite there being 100’s of functional OR genes, knockout mutation of a single gustducin gene results in loss of bitter and sweet taste perception.
2. Wild type mice are averse to salty, sour and bitter substances.
3. The Gα gustducin KO mice fail to recognise bitter substances until they reach relatively high concentrations.
4. Wild type mice are averse to salty and sour substances, but display a preference for sweet tasting substances.
5. The Gα gustducin KO mice are poor at discriminating sweet substances (as well as bitter).

Question 13

Describe experiment that shows Ga gustducin is important for discrimination of bitter and sweet tastes

Answer 13

1. Despite there being 100’s of functional OR genes, knockout mutation of a single gustducin gene results in loss of bitter and sweet taste perception.
2. Wild type mice are averse to salty, sour and bitter substances.
3. The Gα gustducin KO mice fail to recognise bitter substances until they reach relatively high concentrations.
4. Wild type mice are averse to salty and sour substances, but display a preference for sweet tasting substances.
5. The Gα gustducin KO mice are poor at discriminating sweet substances (as well as bitter).

Question 14

How can many odour and taste molecules be interpreted

Answer 14

1. Recognition of thousands of odorant molecules involves conversion of the signal into a common intracellular response via second messengers.
2. In the case of gustatory and olfactory neurons, this signal is relayed directly to the brain.
3. Thus, the connectivity (“wiring”) between sensory cells and target cells processing the signal in the brain is important for recognition of the signal.
4. Common intracellular messengers includes a handful of G proteins and the same simple second messenger cAMP.

Question 15

How can the neuronal wiring be visualised

Answer 15

1. Neuronal wiring visualised by fluorescent protein tracing
2. Labelling of neurons with fluorescent proteins allows tracing from sweet (green) and bitter (red) sensory neurons to distinct parts of the amygdala within the mouse brain.
3. Whole brain fixed and cleared for imaging by light sheet microscopy.
4. Sweet and bitter receptors trace to the anterior basolateral and central, amygdala, respectively.

Question 16

How did knockouts of mice lead to novel aversion

Answer 16

1. Transgenic mice with human-specific receptors acquired novel aversions.
2. KO‘s of specific receptors lead to loss of perception of specific substances
3. Mice expressing bitter substance receptors in “sweet“ neurons resulted in them liking the bitter taste.

Question 17

Describe the receptors in the vertebrate eye

Answer 17

1. Photoreceptors in the retina:
2. Rod cells: light and dark; GPCR rhodopsin, coupled to Gtalpha transducin
3. Cone cells: blue, red, green; GPCR photopsin
4. Due to the nature of the signal, the sensory cells are tucked away at the back eye.

Question 18

Describe the structure of rod photoreceptor cell

Answer 18

1. Outer segment: rhodopsin receptors embedded in membrane discs.
2. Inner segment: organelles, inc. mitochondria.
3. Cell body : contains nucleus
4. Synaptic region: constitutive inhibitory neurotransmitter release
5. The receptors themselves are not presented on the plasma membrane but on stacks of internal membranes.
6. Receptor activation leads to decrease of cGMP levels and hyperpolarisation of the plasma membrane.
7. At the synaptic region, hyperpolarisation blocks release of inhibitory neurotransmitter by rod cell and depolarisation of the postsynaptic cell.

Question 19

Describe structure of receptor

Answer 19

1. 7 transmembrane spanning
2. Both sides of receptor are inside cell
3. Opsin protein is associated in resting state with molecules 11-cis-retinal which is converted to all-trans-retinal when there is light

Question 20

Describe signal transduction in photoreceptors

Answer 20

1. At rest (dark) cGMP-gated Na+ channel allow Na+ influx.
2. Light activates rhodopsin, which in turn activates transducin.
3. Transducin stimulates activity of cGMP phosphodiesterase.
4. As cGMP levels fall, cGMP leaves the Na+ channels on the plasma membrane.
5. The Na+ channels close, causing membrane hyperpolarisation.
6. Note that glutamate released by many photoreceptors inhibits many post-synaptic neurons and stimulates others

Question 21

How is the perception of light amplified

Answer 21

1. Regulation at all levels is necessary to return the cell to a resting state.
2. One rhodopsin molecule absorbs one photon of light
3. Activated receptor can interact with 500 G-Protein transducing molecule
4. Each of these molecules interact 1 to 1 with Cyclic GMP phosphodiesterase molecules
5. 10^5 cyclic GMP molecules are hydrolysed
6. 250 cation channels close
7. 10^6 -10^7 Na+ ions per second are prevented from entering the cell for a period of 1 second
8. Membrane potential is altered by 1 mV
9. Signal relayed to brain

Question 22

How is the signal reversed

Answer 22

1. cGMP-gated Na+ channels are also permeable to Ca 2+ so that Ca 2+ levels also drop in response to light
2. Drop in Ca2+ level activates recoverin, which stimulates guanylyl cyclase activity.
3. recoverin is not a CaM-K and stimulates guanylyl cyclase by association.
4. Regulator of G protein signalling (RGS) proteins associate with active Gα transducin stimulate its GTPase hydrolysis activity
5. Inactivation of transducin prevents further activation of cGMP phosphodiesterase, cGMP levels rise and Na+ channels open.
6. All-trans retinal is released from opsin, transported by a carrier protein to retinal pigmented epithelial cells and converted to 11-cis retinal by a series of enzymatic reactions.

Question 23

What is essential for vision in different light levels

Answer 23

1. Signal adaptation

Question 24

How does the signal adapt to different light levels

Answer 24

1. The activated rhodopsin receptor is recognised by rhodopsin kinase (RK)
2. RK phosphorylates serine/threonine residues on the cytoplasmic portion of rhodopsin, reducing the ability of rhodopsin to activate transducin
3. Rhodopsin is bound by the protein arrestin and becomes fully inactivated
4. the perception of light adaptation is essential to enable sensitivity to be adjusted due different light levels.
5. Otherwise, in going from dim to high light levels the photoreceptor system would be completely overwhelmed.
6. RK is a GRK specific for rhodopsin receptors.

Question 25

Describe vision in deep sea fish

Answer 25

1. Unexpectedly, in addition to having some physical adaptations to the eye, including more low-light sensing rod cells, many stacked on top of each other
2. Instead of the single rhodopsin gene seen in most other vertebrates including other fish, deep sea dwellers have at least 5 and as many as 38
3. These RH1 genes encode opsin proteins optimised to gather the bioluminescent wavelengths emitted by other animals, which are the only sources of light below 1,000m (some of these fish dwell at ove 2,000m) where essentially no residual daylight penetrates.