The Molecular Basis of Dark Vision Ghazwa Aldoori MECP/Cohort 8 Chem. 508 Dr. Michael Topp University of Pennsylvania

Questions Q1. How does the eye recover its sensitivity in the dark following exposure to bright light? Q2. What prevents the regeneration of the vision molecule Rhodopsin in patients with Congenital Stationary Night Blindness? Q 3. What have researchers reached as a possibility of a treatment for this disease?

The sense of vision is a complicated process that requires numerous components of the human eye and the brain to work together. By the end of this presentation, you will be familiar with the following:  The initial step of this powerful sense is carried out in the Retina of the eye. Retina has two kinds of photoreceptor cells, Rods and Cones.  The Fovea, located in the center of the Retina. Responsible for sharp center vision.  Phototransduction: The process of converting absorbed light by rods and cones into electrical signals.  Rhodopsin is the photosensitive pigment (a protein) in the Rod cells of the retina, responsible for the first event in the perception of light.

Detection of light bleaches Rhodopsin into its component parts which then necessitates its regeneration. Dark adaptation: how long it takes to recover vision in dim light. Any conformational change in Rhodopsin disallows its regeneration. One of the causes of conformational changes is the mutation of three amino acids present in this protein. How does this gives rise to Congenital Stationary Night Blindness (CSNB). Further research confirms persistent activation of Rhodopsin even in the absence of light is responsible for Rod cell death. Recent research findings show that gap junctions linking the Rod and Cone cells open and close as regulated by a 24-hour molecular clock located in the Retina.

Retina Contains Cone and Rod cells

Rod Cells Shape of outer segment is rod-like ~120 million rods per retina. Sensitive to dim light. Used in night/scotopic vision Responds to λ max of 498 nm Highest amount in the peripheral region of retina Shape of outer segment is cone-like ~ 7 million cones per retina. Sensitive to colors Three types respond to three different wave peaks: λ max 437nm, λ max 533nm and λ max 564 nm Highest amount in foveal region. Cone Cells

Fovea Located in the center of the Retina. Contains Cone cells only. Figure measures density curves for the rods and cones on the retina Show an enormous density of cones in the fovea centralis. Rod density increases in the peripheral area of the Retina. Approx. 50% of nerve fibers in the optical nerve carries information from the fovea Responsible for sharp center vision (i.e. reading and TV)

Rhodopsin in the Rod Outer segment of the Rod cell Contains membrane bound disks. These disks are densely packed with photo pigment Rhodopsin. Rhodopsin is a member of the super Family of seven-helix, G protein- coupled Receptor protein (Opsin) bound to a Light absorbing chromophore (11-cis retinal). A chromophore is a molecule that can absorb light at a specific wavelength. This 11-cis-retinal chromophore absorbs 1 photon per 200 fs. This one photon is enough to isomerizes11-cis retinal to all trans.

Brief review What happens to the link between Opsin and the chromophore upon isomerization from 11-cis-retinal to all-trans retinal? What essential characteristics of Opsin are necessary for chromophore binding? Schiff Base -Functional group contain a Carbon – Nitrogen double bond. -Nitrogen atom connected aryl or alkyl group but not Hydrogen. Cis–Trans Conformation

Isomerization animation of 11-cis retinal to all trans retinal

Rhodopsin Bleaching It most strongly absorbs green-blue light and therefore appears reddish purple which is why it’s called “visual purple”. Conformational changes triggered by light causes the opsin's absorption spectrum to shift into the UV region, so that the pigment will lose its color and is said to be bleached. Change in shape, size and rigidity of the Chromophore 11-cis-retinal

Photoisomerization: Two Possible Mechanisms 1. Bicycle-Pedal Mechanism: Rotation of two C-H bonds. 2. Hula-twist Mechanism: Proposes rotation of one C-H bond.

Photoisomerization Mechanism “There were, however, theoretical results that suggested that HT is a low-energy pathway (8) and that the minimum- energy pathway of relaxation of an excited conjugated polyene passes through a geometry (conical intersection) identical to that of the HT process” -Robert S. H. Liu, 2002 “Neither the BP mechanism, which would lead to a system with a shifted cis-bond, nor Liu’s Hula-Twist (HT) isomerization model are able to account for the strongly twisted but definitely all-trans-configured geometry observed in the primary Rhodopsin photointermediate.” -Igor Schapiro et al, 2008

Mechanism of Phototransduction Light hits the rod cell and isomerizes retinal. Rhodopsin is converted into metarhodopsin II. Metarhodopsin II activates the G-protein Transducin. The activated α-subunit of Transducin binds GTP (energy molecule) and activates Phosphodiesterase (PDE) PDE catalyzes the hydrolysis of cyclic GMP (cGMP) to GMP cGMP is required to open Na + channels in the plasma membrane. Neuroscience, Purves et al.,2001 cGMP hydrolyzed by PDE reducing its concentration

Phototransduction Is a process by which light is converted into electrical signals in the rod cells of the retina of the eye. Hyperpolarization: is a change in the cell’s membrane potential to make it more negative. It is opposite to depolarization. If a cell has Na + or Ca 2+ currents, then inhibition of those currents will result in a hyperpolarization. But what makes Sodium/Calcium ions open or close? Neuroscience, Purves et al., 2001

Amplification in the phototransduction cascade 1. A single photoactivated rhodopsin catalyses the activation of ~500 transducin molecules. 2. Each transducin can stimulate one phosphodiesterase molecule (enzyme) 3. Each phosphodiesterase molecule breaks down10 3 molecules of cGMP per second Therefore: 4.A single activated rhodopsin causes the hydrolosis of 5x10 5 molecules of cGMP per second. 5. Closing 200 ion channels. 6. That is 2% of the number of channels in the Rod cell. 7. This closure causes a net change in membrane potential of 1mV.

Schiff Base Links 11-cis-retinal to Opsin

Dark Adaptation and Rhodopsin Regeneration Dark adaptation is the amount of time it takes Rhodopsin increase sensitivity to light. The process usually takes about 30 minutes to reach its maximum. Above certain luminance level, the Cone mechanism is involved in mediating vision. Below this level Rod mechanism comes to play mediating night vision.

The Visual Cycle

Congenital Stationary Night Blindness (CSNB) Three times more common in boys than girls. Very difficult to diagnose in children who may communicate the symptoms as simple fear of the dark. Characterized by a prolonged activation of Rhodopsin in the Rod cells and thus continuous activation of the visual cascade. It is hypothesized that this prolonged activation of Rhodopsin leads to Rod cell degeneration (death). No known cure in humans, although gene therapy done on puppies with the disease has restored night vision.

Rhodopsin 3D Structure Primary structure lists the sequence of amino acids in a polypeptide chain. Secondary structure is when this chain fold into a regularly repeated structure. Tertiary structure is the spatial arrangement of the amino acid residues that are far apart in the sequence of the pattern, so it is the interaction of the R groups of the amino acids that determines its 3D structure. The 3D structure of the protein determines its function. Any change in the shape of the structure will affect its function.

Congenital Stationary Night Blindness When one of the following three mutations occurs, human night vision is compromised. A conformational change in Opsin makes it impossible for 11-cis retinal to link, disallowing the response to the absence of light The amino acid mutation of Glycine (G) 90 to Aspartate (D).

The amino acid mutation of Threonine (T) 94 to Isoleucine (I). The amino acid mutation of Alanine (A) 292 Glutamate (E).

Future Although there is not yet a cure for CSNB in humans, three methods hold hope for future patients. Gene Therapy: Recently implemented on dogs with the disease, this method replaces affected dogs’ genes with the genes from healthy dogs, successfully correcting the disorder. Researchers hope to use this procedure’s success to bring relief to humans suffering from the disease. Bionic Eye: Taking advantage of the healthy Optic Nerve in most CSNB patients, this procedure implants a Silicone chip that contains 5000 light sensitive cells in the retina of the patients to replace the malfunctioning Rod cells. It has been successful with other degenerative diseases of the Retina but has yet to be implemented on CSNB patients. As of November, Dr. Mangel and his team of the Ohio State Medical Center discovered that gap junctions linking Rod cells to Cone cells automatically open and close according to a molecular 24-hour clock in the Retina. Dr. Mangel hopes to use this research to gain insight into CSNB and ways to treat/cure it.