wk5-L9-Sensory systems: Phototransduction in Mammals

Light causes hyperpolarization of photoreceptor cells

-real recordings of receptor cells show an increase in magnitude response as the light intensity increases

What is a disadvantage of an inverted retina and how is this resolved?

-A disadvantage of an inverted retina is that light must pass through all retinal layers in order to reach the outer segments that contain the photoreceptors -This issue is resolved as the transparency of the layers prevents the image from degrading -a central high-acuity region prevents light scattering

Does cGMP maintain the resting membrane potential at -40 in the dark?

-In the dark, cGMP conc. within a rod outer segment is high -cGMP binds to cyclic nucleotide gate channels>opening them -permeability of outer segment for cations is high (inc. Na+) -channels are relatively open> cations flow into the cell and keep it depolarized

How does cGMP cause an increase in the opening of sodium channels on photoreceptor cells?

-It activates cGMP-gated ion channels directly(not mediated by PKG) -cGMP-gated ion channel=type of cyclic nucleotide-gated channel -cGMP binding site is found in C-terminal region

What happens after hyperpolarization?

-Photoreceptor cells release the NT glutamate> bind to glutamergic neurons=most common neuron type in the body -In the dark: >photoreceptors are depolarized> glutamate is released -In the light: >photoreceptors are hyperpolarized>less glutamate is released

Mammalian Phototransduction cascade

-Phototransdcution closes cation channels in a rod outer segment. 1-Rhodopsin molecules in disc membrane absorb light and are activated 2-activated rhodopsin stimulates a G-protein(transducin)>activates cGMP phosophodiesterase(PDE) 3-PDE catalyzes the breakdown of cGMP to 5'-GMP 4-The cGMP concentration decreases>cGMP detaches from the cation channels> cation channels close -second messenger-mediate response= decreased Na+ influx>hyperpolarization

What is the function of each of the photoreceptive cells?

-Rods: involved in detection of light during dim light >more sensitive >Nocturnal animals: retina consists mainly of rods -Cones: involved in colour detection during bright light and for high-acuity vision in animals with a fovea >-Diurnal animals: retina consist mainly of cones

What is unusual about the resting membrane potential of the photoreceptor cells?

-The resting membrane potential of a rod/cone is ~ 40mV>more depolarized than the resting membrane potential of a typical neuron -this provides a clue about mechanisms of Phototransduction -this unusual RMP is due to the fact that the resting Na+ permeability of a rod or cone is higher than in normal neuron> membrane potential is less -ve.

Why is the retina inverted?

-The retina is inverted due to the way it develops in the embryo> the more distal layer of a two-layered optic cup

Structure of mammalian eye-focus on retina

-all vertebrates have camera eyes -the cornea and lens focus an inverted image of the visual field on the retina -retina=the photoreceptor containing layer at the back of the eye -the retina of the vertebrate eye is a developmental outgrowth of the brain -impt. part of Phototransduction occurs in the retina -optic nerve sends information to the brain -Fovea= a central high-acuity region in human eye in which intervening cell layers and blood vessels are displaced to the side. A depression of 1.5mm in diameter(5° of visual angle)

cyclic nucleotide gated channels are a type of what channels?

-are ligand gated ion channels that are activated by intracellular ligands(2nd messengers) -ion channels are divided into voltage gated or ligand gated ion channels -ligand gated ion channels can be activated by extracellular/intracellular ligands -cyclic nucleotide gated channels are activated by an intracellular ligand

retinal cGMP-gated ion channels

-belong to family of cyclic nucleotide gated ion channels that are related to voltage-gated ion channels -resemble voltage gated sodium channels in structure -cGMP binding site is formed by the intracellular C-terminal tail

How is the cGMP that keeps the photoreceptor depolarized in the dark generated?

-cGMP is generated by the action of the enzyme guanylyl cyclase -there is a specific form in photoreceptor cells

What generates cGMP in rods?

-cGMP is generated in rods by retinal guanylyl cyclases> RetGC1 an RetGC2 -RetGC1 and RetGC2 are activated by GCAP -In the light: 1-CNG channels close 2- decrease in calcium levels 3-calcium inhibits GCAP> low levels of calcium activate GCAP 4-GCAP activates GCs -In the dark: 1-CNG channels open 2-increase in Ca2+ levels 3-calcium inhibits GCAP> high levels of Ca2+ inhibit GCAP 4-GCs also inhibited

Glutamate effect on bipolar cells

-can cause inhibition(hyperpolarization)/ excitation (depolarization) of postsynaptic bipolar cells -2 types of bipolar cells: 1-ON bipolar cells: express inhibitory metabotropic glutamate receptors(mGluR6)=a GPCR 2-OFF bipolar cells: express excitatory ionotropic AMPA receptors=ligand gated ion channels -nature of receptors expressed on bipolar cells can lead to inhibition/excitation of the postsynaptic cell

Where are the rods and cones found in abundance?

-cones: most abundant in the central fovea>rods area absent -rods: more abundant than cones in the other regions of the retina

Gating of cyclic nucleotide gated channels is voltage dependent -(Marchesi A, Mazzolini M, Torre V-2012)

-cyclic nucleotide gated channels are structurally and evolutionarily related to the voltage gated ion channels -cGMP gated channels evolved from sodium gated channels -have S4-type voltage sensor -cGMP-gated channels of vertebrate photoreceptors are not voltage-gated -This is because: during evolution cyclic-nucleotide gated channels lose their voltage sensing ability when Na+ and K+ permeate> allows them to be open at -ve voltages>necessary for Phototransduction -instead they are activated by increases in intracellular cGMP levels

Why do we look at tiger salamander retina?

-easier to isolate than mammalian and other types of salamander -rod cells and cone cells look like rods and cones under the microscope -most of our knowledge of Phototransduction comes from rods> cuz they are bigger than cones and hence easier to manipulate

What is the downstream effect of rhodopsin in response to light?

-enzymatic breakdown of cGMP> closing of cation channels>hyperpolarization -Breakdown of cGMP> levels of intracellular cGMP go down -cGMP dissociates from cation channels>close -reduction in # of open cation channels -membrane potential becomes more -ve > hyperpolarizing receptor potential

The 5 main branches of GPCRs

-human genome encodes~800 GPCRS that belong to one of 5 main families 1-Glutamate receptors 2-Adhesion receptors 3-Secretin receptors 4-Frizzled/TAS2 receptors 5-Rhodopsin receptors

What is the function of the pigmented epithelium and where is it found?

-it is found at the back of the retina -it's role is to absorb light not captured by the photoreceptors and performs many important metabolic functions>e.g. control of ionic environment around rods and cones and synthesis of a type of retinal

What happens to bipolar cells in the dark?

-photoreceptor(cone/rod) will release glutamate -glutamate inhibits (hyperpolarizes) the ON bipolar cells and excites(depolarizes) the OFF bipolar cells

What happens to bipolar cells in the light?

-photoreceptors release less glutamate -ON bipolar cell loses its inhibition and become active (depolarized) -OFF bipolar cell loses its excitation (becomes hyperpolarized) and becomes silent

Using patch clamping to record ion channel activity in photoreceptors

-possible to record individual currents during AP by using patch clamping technique -provided insight on mechanism of Phototransduction

How is the rhodopsin molecule activated?

-retinal in its conformational state(11-cis) can't activate rhodopsin 1-Light triggers a conformational change from 11-cis to all-trans retinal 2-retinal is intimately associated with opsin>conformational changes in opsin 3-activated rhodopsin is produced -light induced action is rapid> activated rhodopsin is produced in 1ms

Patch clamping of rod outer segment membranes

-rod cell membranes were patch clamped -allowed us to investigate affects of light on membrane response -cGMP increases frequency of sodium channel openings> increases sodium currents in photoreceptor cell membranes. -cGMP=second messenger

Transducin G-protein

-signalling cascade is a classic G-protein pathway -Transducin= a G-protein>like others but unique to Phototransduction pathway -activated by light stimulated rhodopsin (metarhodopsin II) -closely related to G-proteins that mediate metabotropic synaptic actions and chemoreception -activated G-protein stimulates cGMP phosophodiesterase(PDE)

Relationship between sodium gated channels and cGMP gated channels

-structurally similar>same S1-S6 domains -S4 domain is also a voltage sensor -shows that there is a common ancestor between Na +/Ca2+ gated ion channels and cGMP gated channels

PDE and enzymatic degradation of cGMP

-the activated G-protein activates cGMP phosophodiesterase (PDE) -activation of PDE involve dissociation of its catalytic subunit from the regulatory subunit that inhibits its activity -activated PDE decreases the cytoplasmic conc. of cGMP(converts it to 5'GMP)> cation channels close

Retinal cells

-the retina is inverted> photoreceptors in the outermost layer>furthest away from incoming light>towards pigment epithelium -the retina contains: 1-photoreceptor cells: rods and cones 2-a network of neurons: horizontal cells, bipolar cells, amacrine cells, and ganglion cells> perform first stages of visual integration -Locations of cells: >photoreceptors: in an outer nuclear layer at the back of the retina>connected to ganglion cells by bipolar cells> straight through pathway >horizontal cells: form lateral pathway in inner plexiform layer >ganglion cells: output of retina> axons form the optic nerve

Rhodopsin family of GPCRs

-there are many Rhodopsin GPCRs -divided into 4 diff. sub branches: 1-alpha 2-beta 3-gamma 4-delta -alpha-rhodopsin is the prototype for this class>sits between GPCRs mainly involved in Neurotransmission -Opsins are activated by colour photons> enable us to see colour>found in cones -Rhodopsin is found in rods

Summary of Phototransduction

1-activation of rhodopsin by light 2-activated rhodopsin activates G-proteins (transducins) 3-activated transducins lead to stimulation of cGMP-PDEs 4-activated cGMP-PDEs break down cGMP to 5'-GMP 5-intracellular levels of cGMP decrease> cGMP-activated ion channels close 6-number of cGMP-activated ion channels decrease 7-Sodium influx into cell decreases> cell becomes more -ve 8-Hyperpolarization of cell membrane

How is the dark state restored in rods?

1-in the light calcium levels in cytoplasm fall because cGMP gated cation channels close 2-fall in calcium relieves> activation of GCAPs> activation of RetGC 3-RetGC restores cGMP levels>membrane potential is restored to normal~40mv -happens in short timescale to facilitate constant changes on retina

cyclic nucleotides-second messenger molecules

2 main ones: 1-cAMP=cyclic adenosine monophosphate 2-cGMP=cyclic guanosine monophosphate both are related to each other but have slightly diff. pathways

Rhodopsin structure

= a photopigment with 2 parts: 1-retinal(ligand)= the chromophore of animal photoreceptors > exists in two isomers>11-cis and all-trans >attached permanently to a Lys residue in the 7th membrane helix of the opsin protein>buries the retinal deep within the rhodopsin molecule >aldehyde of vitamin A 2-opsin> an integral membrane protein that is attached to retinal -shows 7TM alpha helices characteristic of all GPCRs

cyclic nucleotide-gated channel

= ion channels that are specifically activated by cyclic nucleotides -resemble sodium gated ion channel

cGMP

=cyclic guanosine monophosphate -synthesized from GTP by guanylyl cyclases > not effectors for GPCRs >2 main types of GCs: 1-soluble(activated by NO) 2-membrane(activated by various peptide hormones) -exerts many of its effects in cells by activating protein kinase G(PKG)>phosphorylates various effectors e.g. ion channels -is inactivated by enzymes known as phosphodiesterases(PDEs)>convert cGMP to 5'-GMP

GCAPs

=guanylate cyclase activating proteins -2 types: GCAP1 and GCAP2 -activate RetGC1 and RetGC2 -function= Ca2+ sensors that regulate activity of GCs -high Ca2+ levels=GCAPs inhibited -low Ca2+ levels=active GCAPs> stimulation of RetGC>generation of cGMP

Rhodopsin

=the photosensitive protein in rods -a G-protein coupled receptor -its directly activated by light -7 TM domains -there are 100s of GPCRs in humans and 1000s in C. elegans

Comparison of photoreceptors in dark and light state

Dark: -depolarized outer segment membrane -open cation channels -high level of cGMP in rod cytoplasm Light: -Hyperpolarization of outer segment membrane -closed cation channels -low cGMP levels in cytoplasm

What is the effect of hyperpolarization of photoreceptors on NT release?

If the photoreceptor cells are hyperpolarized there will be a decrease in neurotransmitter release by rods and cones

example of cyclic nucleotide gated ion channels in olfactory receptor neurons-cAMP-gated channels

Olfactory transduction mechanism in cilia membranes of olfactory receptor cells: -many odorants increase levels of cAMP 1-odorant binds to an odorant receptor on the ciliary membrane 2-receptor activates a G-protein 3-G-protein activates Adenylyl cyclase> produces cAMP 4-cAMP binds to and opens a cation channel>allowing influx of Na+ and Ca2+ ions>depolarizes the cell 5-Ca2+ binds to Ca2+ ligand gated chloride channels> allow efflux of Cl->further depolarizing the cell.

Light hyperpolarizes vertebrate photoreceptors

Rod receptor potential: Procedure: -place an electrode inside the rod cell body -shine light upon it Results: -in the light: the rod cell membrane is hyperpolarized -increase in light intensity=increase in magnitude of hyperpolarization Conclusion: -In the dark: retinal rods and cones are relatively depolarized -In the light: retinal rods and cones are hyperpolarized

Vertebrate photoreceptors: general structure of rods and cones

Rods: > divided up into 3 regions: 1-outer segment> cytoplasm filled with intracellular discs (lamellae)> increase SA of light absorbance> transduction into electrical signals 2-inner segment> contains mitochondria>impt. for energy 3-synaptic terminal> where NT is released Cones: -same regions -differences in outer segment> lamellae formed from invaginations of the outer segment PM

Similarities and differences between rods and cones

Similarities: -Both have an inner segment that contains the nucleus,mitochondria and the synaptic terminals -Both have an outer segment that contains ordered lamellae that contains photo pigment molecules -Both have a short ciliary stalk that connects the outer and inner segment>outer segment is derived from the modified cilium -both have lamellae contain the rhodopsin photo pigment Differences: -In rods: ordered lamellae are internalized discs> separated from membrane -in cones: ordered lamellae are invaginations of the outer membrane> stay continuous with outer CM