Gt coupled receptors are activated by light (Rhodopsin activation mechanism)

Gt coupled receptors are activated by light (Rhodopsin activation mechanism)

The rods and cones are the site of transduction of light into a neural signal.

What exactly happens at the molecular level?

  • Visual stimuli cause changes in the photo pigment molecule that lead to changes in membrane potential of the photoreceptor cell.

Over all steps involved in rhodopsin signal transduction

Figure 1: Molecular consequences of photon absorption by rhodopsin in the outer segment

Step 1: Light absorption and activation of rhodopsin

  • Visual transduction begins when light falls on Rhodopsin.
  • Light triggers conformational changes in the receptor rhodopsin.
  • Thousands of molecules of rhodopsin are present in each disk of the outer segments of rod cells.
  • When a photon is absorbed by the retinal component of rhodopsin, the energy causes a photochemical change. 11-cis-retinal (bent form) is converted to all trans– retinal (linear isomer).
  • Retinal is a hydrocarbon molecule related to vitamin A.
  • When a photon hits retinal, the long hydrocarbon chain of the molecule is biochemically altered.
  • Before interacting with a photon, retinal’s flexible double bonded carbons are in the cis form This molecule is referred to as 11-cis-retinal.
  • Photons cause some of the double bonded carbons within the chain to switch from a cis to  trans conformation (straight hydrocarbon chain). This process is called photo isomerization.
  • This change in the structure of the chromophore causes conformational changes in the rhodopsin molecule.
  • This isomerization of retinal activates the rhodopsin, starting a cascade of events that ends with the closing of Na+ channels in the membrane of the photoreceptor.
Figure 2:Retinal Isomers (The retinal molecule has two isomers, (a) one before a photon interacts with it and (b) one that is altered through photo isomerization).

Metarhodopsin ӀӀ (activated rhodopsin)

  •  Opsin is covalently bound to all-trans-retinal is called metarhodopsin II, or activated opsin.
  • Metarhodopsin II  excites the electrical changes in rods and the rods then transmit the image to CNS through optic nerve. This process is called Phototransduction.
Figure 3: Pathway showing light induced activation of rhodopsin

Step 2: Activated rhodopsin activates the transducin coupled to the receptor

  • Metarhodopsin II initiates the visual photo transduction pathway by stimulating the G protein transducin (Gt).

In the dark:

  • GDP is bound to the G subunit (inactive form).
  • All three subunits of the protein (Tα, Tβ and Tϒ) remain together and no signal is sent.

 When rhodopsin is excited by light:

  • Alpha subunit replaces bound GDP by GTP (active form).
  • Transducin then dissociates into Tα and Tβϒ.
  • The Tα – GTP carries the signal from the excited receptor to the cGMP phosphodiesterase enzyme (PDE).

Step 3: Activation of cGMP phosphodiesterase enzyme (PDE) by the G– GTP subunit.

cGMP phosphodiesterase enzyme

  • The cGMP-specific PDE is unique to the visual cells of the retina
  • cGMP phosphodiesterase is the effector protein for Gt.

Structure:

  • an oligomer with α, β and two inhibitory γ subunits.
  • PDE is an integral protein with its active site on the cytoplasmic side of the disk membrane.

 Mechanisms for the activation of PDE:

  • In the dark: Tightly bound inhibitory subunit (ϒ subunit) very effectively suppresses PDE activity.
  • The free Gt α GTP complex that is generated after light absorption by rhodopsin binds to the two inhibitory ϒ subunits of cGMP phosphodiesterase.
  • It results in the releases of the PDE γ subunit from the catalytic subunits (α and β subunit) and catalytic units are activated. The enzyme’s activity immediately increases by several orders of magnitude.
  • This is another example of how signal-induced removal of an inhibitor can quickly activate an enzyme, a common mechanism in signaling pathways.

Step 4: Reduction in the level of cGMP in the outer segment of rods

  • Each molecule of active PDE degrades many molecules of cGMP to the biologically inactive 5’- GMP lowering [cGMP] in the outer segment within a fraction of a second.

cGMP phosphodiesterase action: catalyses the conversion of cGMP to 5’ GMP.

This enzyme belongs to the family of hydrolases, specifically those acting on phosphoric diester bonds.

Step 5: Closing of cation channels and membrane hyper polarization

  • The key transducing molecule linking activated opsin to the closing of cation channels in the rod-cell plasma membrane is the second messenger cyclic GMP (cGMP).
  • The high level of cGMP present in the dark acts to keep cGMP gated cation channels
  • In the dark, the membrane potential of a rod cell is about – 30 mV, considerably less than the resting potential (-60 to – 90 mV) typical of neurons and other electrically active cells.
  • As a consequence of this depolarization, rod cells in the dark are constantly secreting neurotransmitters (Eg: glutamate) and the bipolar inter neurons with which they synapse are continually being stimulated.
  • The depolarized state of the plasma membrane of resting rod cells is due to the presence of a large number of open nonselective ion channels that admit Na + and Ca2+ as well as K+.
  • Absorption of light by rhodopsin leads to a decrease in the level of cGMP which in turn causes the closure of cGMP gated ion channels.
  • This will block the re entry of Na+ and Ca2+ into the outer segment and hyperpolarizing the membrane of the rod or cone cell.
  • Closing of these channels causes the membrane potential to become more negative.
  • When more photons are absorbed by rhodopsin, more channels are closed and fewer Na + ions cross the membrane from the outside. The membrane potential becomes more negative and less neurotransmitter is released into the outer synaptic layer of the retina.
  • The hyperpolarized membrane does not release glutamate to the bipolar cell.
  • Activated neurons stimulate ganglion cells, which send action potentials via the optic nerve.
  • This change is transmitted to the brain where it is perceived as light.

(The neurotransmitter in rods and cones is the amino acid glutamate (glutamic acid). At synapses between rods and some bipolar cells, glutamate is an inhibitory neurotransmitter:It triggers inhibitory postsynaptic potentials (IPSPs) that hyperpolarize the bipolar cells and prevent them from sending signals on to the ganglion cells).

  • A single photon absorbed by a resting rod cell produces a measurable response, a decrease in the membrane potential of about 1 mV, which in amphibians lasts a second or two.
  • Humans are able to detect a flash of as few as five photons.

(Through this process, the initial stimulus (a photon) changes the Vm of the cell).

Thus, unlike most other sensory neurons (which become depolarized by exposure to a stimulus), visual receptors become hyperpolarized and are driven away from the threshold.

Figure 4: Operation of rod photoreceptors (Light causes a hyperpolarizing receptor potential in photoreceptors, which decreases release of an inhibitory neurotransmitter glutamate).

Step 6: Reduction in the level of cytosolic Ca2+ions

  • Calcium levels drop during illumination, because the steady state Ca2+ in the outer segment is the result of outward pumping of Ca2+ through the Na+ – Ca2+ exchanger of the plasma membrane and inward movement of Ca2+ through open cGMP gated channels.
  • In the dark, this produces a Ca2+ concentration of about 500 nM which is enough to inhibit cGMP synthesis.
  • After brief illumination, Ca2+ entry slows and Ca2+ level declines.

Step 7: Reduction in Ca2+ level leads to the activation of guanylyl cyclase enzyme

  • When the cytosolic Ca2+ concentration decreased, the inhibition of guanylyl cyclase by Ca2+ is relieved, and the cyclase converts GTP to cGMP to  return cGMP concentration to its “dark” level. Finally the cation channels re open and return the Vm to pre stimulus level.
  • Rod outer segments contain an unusually high concentration (≈0.07 mM) of cGMP, which is continuously formed from GTP in a reaction catalyzed by guanylyl cyclase that appears to be unaffected by light.

Step 8: Rhodopsin desensitization by Phosphorylation

  • Rhodopsin itself also undergoes changes in response to prolonged illumination.
  • The conformational change induced by light absorption exposes several Thr and Ser residues in the carboxyl-terminal domain.
  • These residues are quickly phosphorylated by rhodopsin kinase which is functionally and structurally homologous to the β – adrenergic kinase (βARK) that desensitizes the β – adrenergic receptor.
  • The Ca2+ binding protein recoverin inhibits rhodopsin kinase at high (Ca2+).
  • But the inhibition is relieved when Ca2+drops after illumination.
  • The phosphorylated carboxyl terminal domain of rhodopsin is bound by the protein arrestin 1, preventing further interaction between activated rhodopsin and transducin.

Rod Cells Adapt to Varying Levels of Ambient Light

Figure 5: Role of opsin phosphorylation in adaptation of rod cells to changes in ambient light levels.

Visual adaptation:  involves phosphorylation of activated opsin (O*) by rhodopsin kinase

  • Each opsin molecule has three principal serine phosphorylation sites.
  • Light-activated opsin (O*), but not dark-adapted rhodopsin, is a substrate for rhodopsin kinase.
  • The extent of opsin phosphorylation is directly proportional to the amount of time each opsin molecule spends in the light activated.
  • The ability of O* to activate Gt is inversely proportional to the number of phosphorylated residues.
  • Thus the higher the ambient light level, the greater the extent of opsin phosphorylation and the larger the increase in light level needed to activate the same number of Gt (transducin) molecules.
  • At very high light levels, protein beta arrestin binds to the C-terminal segment of opsin. The bound beta arrestin prevents interaction of Gt with O*, totally blocking formation of the active Gtα GTP complex and causing a shutdown of all rod-cell activity.

The Visual Signal Is Quickly Terminated

  • Very shortly after illumination of the rod or cone cells the photo sensory system shuts off.
  • Activated opsin is unstable and spontaneously dissociates into its component parts, releasing opsin and all trans– retinal, thereby terminating visual signaling.
  • α subunit of transducin (with bound GTP) has intrinsic GTPase activity.
  • Conversion of active Gtα GTP back to inactive Gtα GDP is accelerated by a GTPase activating protein (GAP) specific for Gtα
  • Within milliseconds after the decrease in light intensity, GTP is hydrolyzed and Tα reassociates with Tβϒ.
  • The inhibitory subunit of PDE, which had been bound to Tα – GTP, is released and reassociates with PDE, strongly inhibiting that enzyme.

In mammals Gt normally remains in the active GTP-bound state for only a fraction of a second.

Thus cGMP phosphodiesterase rapidly becomes inactivated, and the cGMP level gradually rises to its original level when the light stimulus is removed.

Step 9: Regeneration of rhodopsin from Metarhodopsin II

  • Rhodopsin pigment must be regenerated for further photo transduction to occur.
  • Rhodopsin is reformed by conversion of all-trans retinal to 11-cis retinal in presence of enzyme retinal isomerase.
  • Once formed 11-cis retinal automatically combines with scotopsin to form rhodopsin which remains stable until it is decomposed by absorption of photon.
  • The bleaching of the rhodopsin takes place in the presence of light, whereas the regeneration event occurs in the absence of light.

Figure: The cyclical bleaching and regeneration of photopigment (Blue arrows indicate bleaching steps; black arrows indicate regeneration steps).

Amplification of the Visual Signal Occurs in the Rod and Cone Cells

  • Several steps in the visual-transduction process result in great amplification of the signal.
  • Each excited rhodopsin molecule activates at least 500 molecules of transducin, each of which can activate a molecule of PDE.
  • This phosphodiesterase has a remarkably high turnover number, each activated molecule hydrolyzing 4,200 molecules of cGMP per second.
  • The binding of cGMP to cGMP gated ion channels is cooperative (at least three cGMP molecules must be bound to open one channel), and a relatively small change in [cGMP] therefore registers as a large change in ion conductance.
  • The result of these amplifications is exquisite sensitivity to light.
  • Absorption of a single photon closes 1,000 or more ion channels and changes the cell’s membrane potential by about 1 mV.

Photo bleaching

Definition: loss of colour by a pigment (such as chlorophyll or rhodopsin) when illuminated.

  • When rhodopsin is exposed to light, it immediately photo bleaches (trans-retinal completely separates from opsin. The final products look colorless).
  • In humans, it is regenerated fully in about 30 minutes.
  • Until the retinal molecule is changed back to the 11-cis-retinal shape, the opsin cannot respond to light energy.
  • In normal sunlight, rhodopsin will be constantly bleached while the cones are active.
  • In a darkened room, there is not enough light to activate cone opsin and vision is entirely dependent on rods.

Additional information:

Humans cannot synthesize retinal from simpler precursors and must obtain it in the diet in the form of vitamin A.

Dietary deficiency of vitamin A causes night blindness (poor vision at night or in dim light).

 

 

 

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