Lecture 12 - Signalling pathways in practice Flashcards
Describe how the olfactory system is involved in sensory perception
Many mammals and other organisms can distinguish between thousands of different smells (thousands of different odorant molecules) in our olfactory systems with a great deal of specificity. Therefore, the pathways involved need to be highly tuned to discriminate between different smells. Odorants are generally small, organic, volatile molecules.
They are volatile because they can be volatilized, and those volatile molecules can move into the nasal passages to where the molecular machinery which is responsible for the perception of those molecules is located. When you are smelling things, you are detecting small molecular quantities of whatever it is you are smelling.
It is the shape of the odorant molecule which appears to be important in the initial detection of those molecules. The shape of the molecules is important because it allows the interaction with the receptors which are specific for those different molecules.
Describe olfactory perception
Volatile compounds enter the nasal cavity where the main olfactory epithelium is located and are innervated back to the olfactory bulb. Importantly, the sensory neurons have 12 cilia which project into the mucosal lining of the nasal cavity which contain all the molecular machinery which is responsible for the perception of different odorant molecules triggering a signal that goes back to the olfactory bulb and is detected in the brain.
Describe oderent receptors
This signaling starts off with activation of a G protein coupled receptor. The odorant receptors conformed to the basic G protein coupled receptor motif in that they have 7 trans membrane spanning Alpha helices. They have several extracellular domains and cytosolic domains and at general level their structure is conserved across the odorant receptors. However, there are many different genes, >1000, producing odorant receptors and the amino acid sequence varies considerably between the different odorant receptors.
Describe the oderant perception signaling pathway
Odorant perception signaling pathway.
1. The odorant interacts with a high degree of specificity with a particular G protein coupled receptor via specific amino acid residues on different alpha helices.
2. This brings about a conformational change in the receptor that allows the signal to be transduced from the extracellular environment across, the plasma membrane, and into the cytosol and then to activate downstream signaling.
3. The first signaling intermediate activated is a heterotrimeric G protein called G-olf (G olfactory).
4. This results in the exchange of GDP bound to the guanyl nucleotide binding site on the alpha of the G protein for GTP which activates the G protein allowing the Alpha subunit carrying GTP to diffuse from the beta gamma complex.
5. It is the Alpha subunit which is important in activating downstream signaling although there are instances where the Beta-gamma complexes can be very important in signaling too. The Alpha subunit with GTP in the guanyl nucleotide binding site can then activate the next downstream effector which is an adenylate cyclase.
6. Once activated, the adenylate cyclase enzyme converts ATP in to the second messenger cyclic AMP causing a very rapid increase in the concentrations of cyclic AMP in the cytosol which peaks very in about 100 milliseconds.
7. cyclic AMP then targets the next downstream effector.
8. The odorant signaling pathway targets an ion channel; specifically, a cyclic AMP-gated cation channel. The increase in cyclic AMP gates the cyclic AMP-gated channel open activating the channel and allow cations, specifically sodium and calcium, to enter the cell.
9. Therefore, there is the movement of positive ions from outside the cell to the inside of the cell so that the inside of the cell gets more positive. This results in depolarization of the plasma membrane.
10. The increase in calcium, in addition to contributing to the positive charge inside the cell, can also act as a second Messenger activating another channel in the plasma membrane, this time it’s a chloride efflux channel.
11. Activation of this channel allows the efflux of negative ions which again makes the inside of the cell more positive contributing to the depolarization of the plasma membrane.
12. Therefore, each of these steps causes membrane depolarization and that membrane depolarization will be sufficient to activate a voltage-gated sodium channel in the cell bodies and that will trigger the action potential, which is then perceived as that odorant perception.
What is the function of human rod and cone cells.
Photodetection takes place in the retina. There are two types of photodetection cells in the retina: cones cells are typically better at detecting bright light and colour; rod cells which are better at detecting low levels of light and which are therefore better at perceiving black and white.
There are ~90 million rod cells in the human retina. They are made up of two basic components: the inner segment and the outer segment. The inner segment is not involved directly in photodetection but is more involved in metabolism and housekeeping for that rod cell. It is the outer segment which is important and which contains the molecular machinery responsible for photodetection. The outer segment of the rod cells contains a stack of ~1000 membrane discs. The molecular machinery responsible for the perception of light in these cells is distributed between the membranes of those disks and the plasma membrane of the rod cell.
Describe the light induced hyperpolarisation of rod cells
The membrane potential of a rod cell changes during light perception due to changes in the gating of a cyclic GMP-gated channel in the plasma membrane. In an unstimulated cell, unstimulated by light, the resting potential of the cell is about -30 millivolts. Typically, the resting membrane potential of a cell will be -30 to -90 millivolts. Therefore, in an unstimulated resting cell, the plasma membrane is depolarized, i.e., less negative. This is because the plasma membrane cyclic GMP gated channel, which is an anion channel, is open, allowing the influx of positive ions into the cell.
Closure of the cyclic GMP-gated channels in the rod cell plasm membrane results in a reduced influx of positive ions, sodium and calcium, reducing the buildup of positive ions on the inside of the plasma membrane so that the membrane potential of the cell becomes more negative, i.e. it moves to be hyperpolarized reducing the production of the neurotransmitter glutamate, and that reduced production is perceived as that light stimulus.
What is rhodopsin?
Light is perceived by rhodopsin in rod cells, which is analogous to a typical G protein coupled receptor in structure. It has the seven transmembrane spanning Alpha helices, but rather than having a ’ligand binding pocket’ in the centre of that barrel-like structure formed by those Alpha helices it has the photosensitive pigment which undergoes isomerization in response to light. In the unstimulated form the photoreceptive moiety is in the form of 11-cis-retinal linked to Opsin which sits within that barrel light structure of the G Protein coupled Receptor structure. Light results in cis-trans isomerization of the retinal moiety through isomerization of double bonds in that moiety to produce an all-trans-retinal moiety activating the Opsin. This activates the photoreceptor which is then termed meta-rhodopsin II. This is analogous to the activation of a classic G protein coupled Receptor which is responsible resulting in a change in conformation of the receptor allowing it to interact with downstream signaling elements.
Describe the light perception signalling pathway.
The initial molecular machinery responsible for perception of the light it is located in the membrane of the disks in the outer segment of the rod cell. Light-induced isomerization of rhodopsin into Meta-rhodopsin II, brings about a conformational change that allows the receptor to interact with and activate a G protein, a heterotrimeric G protein, called transducin.
This allows the exchange of GDP replacing it with GTP on the Alpha subunit, activating the heterotrimeric G protein allowing the Alpha subunit with GTP in the guanyl nucleotide-binding site to dissociate from the beta-gamma complex.
The activated transducin Alpha-GTP subunit can then diffuse to and activate the next downstream effector in the signaling pathway, which is an enzyme called phosphodiesterase, which is again located with the disk membrane.
This removes an inhibitory subunit from the phosphodiesterase and in doing so activates the phosphodiesterase. Rather than leading to an increase in the concentration of a second messenger, activation of phosphodiesterase allows the enzyme to hydrolyze cyclic GMP back to GMP therefore reducing the concentration of the second messenger cyclic GMP in the cytosol rather than as with adenylate cyclase and cyclic increasing the second messenger concentration.
The decrease in the second messenger cyclic GMP is important because that can influence the activity of downstream effectors, not in the disk membrane, but effectors located in the plasma membrane of the rod cell. Specifically, the cyclic GMP gated anion channel which is gated open by cyclic GMP in the unstimulated rod cell due to the presence of high levels of cytosolic cyclic GMOP allowing positive sodium (and calcium) ions to enter the cell making the inside of the membrane more positive resulting in membrane depolarization from -60 to -90 millivolts to only -30 millivolts allowing the continuous production of the neurotransmitter glutamate. However, activation of the phosphodiesterase results in a reduction of cytosolic cyclic GMP, removing cyclic GMP from the cyclic GMP-gated channels, and therefore closing those channels.
Light induced hyper-polarization of rod cells
Closure of the cyclic GMP-gated channels in the rod cell plasm membrane results in a reduced influx of positive ions, sodium and calcium, reducing the buildup of positive ions on the inside of the plasma membrane so that the membrane potential of the cell becomes more negative, i.e. it moves to be hyperpolarized reducing the production of the neurotransmitter glutamate, and that reduced production is perceived as that light stimulus.
Describe calcium AB signalling in guard cells.
Stomata are pores on the surface of leaves the allow gas exchange. Stomatal guard cells are a pair of specialized cells that surround the pore and which control the size of the pore through changes in the water content (turgor) of these cells. The water content is controlled by the levels of osmotically active ions the most important of which are potassium and it’s two main counter ions chloride and malate. To open, there needs to be an increase in the osmotically active ion content; water then follows in, and the cells swell and the stomata open. To close, they lose, or break down in the case of malate, the osmotically active ions; water follows, the cells shrink, and the stomata shut down. These movements of ions occur in response to many different stimuli, so the ion channels that regulate their movements have to be tightly controlled through to bring about an appropriate change in aperture, an appropriate physiological response, to a given set of environmental conditions, i.e. a given set of stimuli. This involves complex signaling pathways in which calcium is a second messenger.
Describe the effect of ECTA on ABA-induced stomatal closure.
Effect of Removing External Calcium:
Using ETGA reduces ABA’s impact on stomatal aperture, highlighting calcium signaling’s role in regulating ion channels controlling stomatal aperture.
ABA-Induced Stomatal Closure Mechanism:
ABA promotes osmotically active ion efflux, reducing cell concentration, and inducing water flow, leading to cell collapse and stomatal closure.
Key ion channels involved:
Plasma membrane channels: potassium influx (K+in), anion efflux (ICl), potassium efflux (K+out).
Vacuolar membrane channel: potassium efflux (TPK1).
Role of Cytosolic Calcium Increase Induced by ABA:
ABA triggers cytosolic calcium rise, activating calcium-dependent protein kinases.
Effects include:
Inhibition of K+in, preventing stomatal opening.
Activation of anion efflux channels, allowing ion efflux.
Promotion of TPK1 and K+out activation, causing stomatal closure.
Amplification of Calcium Signal:
Sequential depolarization of plasma membrane and calcium-induced calcium release magnify calcium signal.
Calcium-mobilizing second messengers like IP3, cyclic ADP ribose, and sphingosine-1-phosphate further amplify calcium signaling.
