Photosynthesis Proton Pumping Vision

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PhotosynthesisProton PumpingVision
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Spectroscopic studies of ultrafast electron
transfer in photosynthesis
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Dynamics in the Photosynthetic Reaction Center
(RC)

• Charge separation and recombination are not
  temperature dependent.
• Dielectric symmetry breaking allows charge
  separation down one of two symmetry related
  branches.
• The quantum yield for charge separation is 1.
• The quantum for primary charge separation is
  electric field dependent.
  

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Light Harvesting by Energy Transfer
Trapping by charge separation
Absorption of light
BChl BChl BChl BChl P
RC
Antennae
The scheme above depicts energy transfer among
the antennae until the energy is ultimately
trapped by the Reaction Center. Rapid electron
transfer prevents the energy from returning to
the antennae.
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Structure of the Photosynthetic Reaction Center
of Rb Sphaeroides
Periplasm
Membrane
Cytoplasm
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Chromophores in the Bacterial Photosynthetic
Reaction Center
The special pair The BChl dimer The primary
donor P
Accessory BChl B
C2
Bpheo H
Ubiquinone QA
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Chromophores in the Bacterial Photosynthetic
Reaction Center
The special pair The BChl dimer The primary
donor P
Accessory BChl B
Bpheo H
Ubiquinone QA
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A reaction scheme for charge separation and
recombination
P is excited by light. The primary charge
separation steps are not temperature
dependent. The charge recombination process kR
occurs in RCs with no QB.
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Definitions of electron and energy transfer
processes
We define three electron transfer
processes Charge separation DA DA- Charge
shift D-A DA- Charge recombination DA-
DA Energy transfer is the transfer of
excitation energy from one molecule to
another. Excitation transfer DA DA
Donor (D) Acceptor(A)
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Rate constant for energy transfer and electron
transfer
V is the electronic coupling. V is proportional
to the overlap of reactant and product
electronic wavefunctions. FC is the
Franck-Condon factor. FC is the square of the
overlap of nuclear wavefunctions.
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Diabatic electronic coupling
The hamiltonian is the nuclear momentum
operator. This operator mixes the orthogonal
DA and DA- states in the diabatic
representation.
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Adiabatic electronic coupling
In the adiabatic representation the two states
are not orthogonal so the electronic coupling
is proportional to their overlap at the crossing
point.
The two representations are equivalent!
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The Franck-Condon factor
In electron transfer and energy transfer there
can be a barrier to the reaction. The barrier
height depends on the change in nuclear
displacement, D in the product state.
Larger displacement leads to a higher barrier
Reactants Products
D
D
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The FC factor in absorption, energy, and electron
transfer are all the same
Energy from a photon must be supplied to permit
this transition.
Here we compare a product state that is lower in
energy and one that is much higher in energy than
the reactants.
D
Reactants Products
D
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The FC factor can be expressed as a function of
the energy gap and reorganization energy
e energy gap l reorganization energy
l
e
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The barrier height changes as a function of the
energy gap
e gt l
E
e l
E 0
e lt l
E
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Marcus theory predicts a Gaussian dependence of
rate on the energy gap
The FC factor is
The barrier is zero when l e. This form of
rate theory applies only for coupling of low
frequency solvent modes.
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The rate vs. free energy curve is a non-radiative
spectrum for the ET process
Normal
Inverted
Activationless
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Absorption spectrum of the RC
P is broader than B. P is shifted to the red.
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Spectroscopic observation of the primary charge
separation
Stimulated Emission
Electrochromic Shift of B
Bleach of P
Kinetics of electron transfer t 3.5 ps
- - - 30 ps ____ 0.5 ps
0 10 20 30 40
800 900
Time (ps)
Wavelength (nm)
Murchison et al. Biochemistry 1993, 32, 3498
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The small fluorescence quantum yield from P
arises from the competition of electron transfer
The fluorescence yield fromBChl monomer in
solution is 0.10 due to the much slower
non-radiative decay of the monomer.
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Hydraulic analogy for quantum yield
Tank with two drains
kA
kB
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The primary charge separation rate is the most
efficient electron transfer process known
The quantum yield is nearly one. The
edge-to-edge distance of P and H is 10 Å. The
charge separation occurs only down one branch
of the pigment molecules.
P
H
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Fundamental relationship between transition
moment polarizability and state-to-state
difference polarizability
The transition polarizability can be
written kIC(F) kIC(1 AF BF2) and kIS(F)
kIS(1 AF BF2) The terms are obtained from
the above definitions
Franzen et al. J. Phys. Chem. submitted
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Da is known from experiment
From low temperature electroabsorption we know
that Da is huge for the primary donor. Therefore
we can estimate the transition dipole
moments. The resulting model is consistent with
a large increase in rate constant for
non-radiative decay (both singlet and triplet)
due to admixture of CT states.
Franzen et al. J. Phys. Chem. submitted
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Admixture of a CT state produces a greater
excited state displacement
Pure Exciton
Mixed CT
D 2.45
D 0.45
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Excitation wavelengths for a resonance Raman
comparison of P and B
____ Normal RC ---- P oxidized The P
band bleaches if P is oxidized to P. This can be
done chemically using Fe(CN)63-.
Raman Excitation Wavelengths
B
P
H
Shreve, Franzen et al. PNAS 1991, 88, 11207
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P has strongly coupled low frequency modes


P
lexc 850 nm
Spectrum A Reconstruction of SERDS Spectrum
B Intradimer modes that are not found in
B. Spectrum C Fe(CN)63- control
experiment proves that these modes belong to P.
B
lexc 810 nm


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Stimulated emission in the wavepacket picture
Stimulated emission from a
vibrationally coherent state
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Excited state coherent vibrational motion in wild
type and DLL
DLL
WT
Franzen and
Martin Ann. Rev. Phys. Chem. 1995, 46, 453
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Bacteriorhodopsin

• The proton pump
• The photocycle
• Biological computing
• Raman spectroscopy

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The concept of a proton pump
Photosynthesis in bacteria and plants results in
proton pumping. The energy is used to
phosphorylate ADP (ADP P ? ATP). ATP is the
main energy carrying molecule of the
cell. Bacteriorhodopsin is a proton pumping
protein found in archaea.
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Bacteriorhodopsin is a photosynthetic pigment
used by archaea, most notably halobacteria. It
acts as a proton pump, i.e. it captures light
energy and uses it to move protons across the
membrane out of the cell. The resulting proton
gradient is subsequently converted into chemical
energy. Bacteriorhodopsin is an integral membrane
protein usually found in two-dimensional
crystalline patches known as "purple membrane",
which can occupy up to nearly 50 of the surface
area of the archaeal cell. The repeating element
of the hexagonal lattice is composed of three
identical protein chains, each rotated by 120
degrees relative to the others. Each chain has
seven transmembrane alpha helices and contains
one molecule of retinal buried deep within. It
is the retinal molecule that changes its
conformation when absorbing a photon, resulting
in a conformational change of the surrounding
protein and the proton pumping action.
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The bacteriorhodopsin molecule is purple and is
most efficient at absorbing green light
(wavelength 500-650 nm, with the absorption
maximum at 568 nm). The three-dimensional
tertiary structure of bacteriorhodopsin
resembles that of vertebrate rhodopsin, the
pigments that sense light in the retina.
Rhodopsins also contain retinal, however the
functions of rhodopsin and bacteriorhodopsin are
different and there is no homology of their
amino acid sequences. Both rhodopsin and
bacteriorhodopsin belong to the 7TM
receptor family of proteins, but rhodopsin is a
G-protein coupled receptor and bacteriorhodopsin
is not. In the first use of electron
cryo- crystallography to obtain an atomic-level
protein structure, the structure of
bacteriorhodopsin was resolved in 1990. It was
then used as a template to build models of other
G protein- coupled receptors before
crystallographic structures were also available
for these proteins.
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• Type 1. Transmembrane (2 classes)
• Class 1.1 Alpha-helical transmembrane (39
  superfamilies)
• Superfamily 1.1.01 Rhodopsin-like proteins (2
  families)
• Family 1.1.01.01 Ion-translocating microbial
  rhodopsin (8 proteins)
• Species Halobacterium salinarium (3 proteins)
• Localization Archaebacterial membrane (16
  proteins)

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Simplified schematic of how bacterio- rhodopsin
works Incoming light is converted to create a
charge (pH) difference across the cell membrane
(symbolized by darker green). At this level of
detail we can see that The amino acids in a
protein channel assist in moving the H ions
from one side to the other. The driving force
is the change in coformation of the retinal
(shown in red) and the energy source for the
structural changes is the absorption of light.
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The formation of the Schiff base
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The photocycle
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The photocycle
The photocycle is initiated by the absorption of
a photon by the retinal co-factor (Amax at 568
nm), which is linked to the protein by a
protonated Schiff base at Lys216.  Photon
absorption causes rapid rearrangment of the
electronic structure of the extended conjugated
retinal p-system which ultimately results in
all-trans to 13-cis isomerization at the C13C14
double bond (K intermediate).  This
isomerization in turn reduces the proton
affinity of the charged Schiff base nitrogen,
which loses its proton to the initial acceptor
group, Asp85, probably via a water molecule (M
intermediate Amax 410 nm).  Concurrently, a
proton is released on the extra-cellular side of
the membrane, most probably from glu-204 with
the help of arg-82.   Subsequently, in the N
intermediate, the schiff base is reprotonated
from asp-96, which is itself reprotonated from
the cytoplasmic side of the membrane. In the O
intermediate, the retinal reisomerizes to an
all-trans configuration, while the proton release
group glu-204 is reprotonated by asp-85.
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Changes at Cryogenic Temperature

• At around the temperature of 80 K, the native
  protein undergoes
• this photocycle and switches between a green
  absorbing state and
• a red absorbing state. At room temperature, the
  protein switches
• between a green absorbing state and a blue
  absorbing state. In both
• the ground (green) and excited (red or blue)
  states, the chromophore
• displays several metastable configurations. The
  main event follows
• these steps
• A change in the shape of the conformational
  potential energy
• surface resulting from electron excitation
• 2. A conformational change
• 3. A non-radiative decay to the ground state
• The single critical step in the proton pumping
  ability of the protein
• is the transfer of the Schiff base proton to D85
  in the L ? M reaction.

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The photocycle at room temperature
In the L state, the Schiff base exhibits strong
H-bonding with close water molecules and
distorts the chromophore near the Schiff base.
These water molecules are bound to the anionic
Asp85 and Asp212. These events coincide with an
interaction of Trp182 with the retinal skeleton
by the 9-methyl group. These events bring about
the deprotonation of the Schiff base. Also in
the L intermediate state, the backbone has good
local structural flexibility. This is evidenced
by the many different change in the peptide C to
O double bond stretching vibrational frequencies.
Some of these frequency variations correlate to
the O to H single bond stretching vibrational
frequencies. This indicates that the structural
changes can come from changing interaction with
water molecules. A network of H-bonding
including bonds between water and peptides,
exists between two pieces of the protein, Asp85
and Asp96. This network exhibits changes most
often in the bR to L transformation, which would
be the first step in writing to a block of
bacteriorhodopsin memory.
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Biological computers
The relative stability of some of the
intermediate states determines their usefulness
in computing applications. The initial state of
the native protein, often designated bR, is
quite stable. Some of the intermediates are
stable at about 80K and some are stable at room
temperature, lending themselves to different
types of RAM. For computers, the two or three
most stable states of the protein would be used
to record data in binary form. This is the
proposed photocycle for computing needs. Two
states of bR are shown in the cuvettes at the
right.
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Wavepacket dynamics Absorption
i(t)ñ eiHtiñ
áii(t)ñ
ái
v 0
D
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Fourier transform of exponentially damped
sinusoid is a Lorentzian
á00ñ
FT D 1
á01ñ
ái
v 0
D
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Wavepacket dynamics Raman
i(t)ñ eiHtiñ
áfi(t)ñ
áf
v 1
ái
v 0
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FT of áfi(t)ñ yields the REP
á00ñá01ñ á01ñá11ñ
FT
áf
ái
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Raman difference spectrum for bR
Probe only
Excitation 514 nm Probe 676 nm The K spectrum
is a transient spectrum.
Pump and probe
Difference (Intermediate)
HOOP
C-H wags
Raman shift (cm-1)
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Isotopic labeling studies (K and model system)
The HOOP (vinyl-H out-of-plane) Modes are not
enhanced for either planar structure. The
planar models 13-cis and all-trans are
protonated Schiff bases.
HOOP
Raman shift (cm-1)
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Physical Chemistry of Vision

• The eye
• Retinal
• Rhodopsin
• The photocycle
• Photopigments and color
• The generation of nerve impulses

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The eye

• The cornea
• admits light to the interior of the eye and
• bends the light rays to that they can be brought
  to a focus.
• The retina
• The retina is the inner layer of the eye. It
  contains the light
• receptors, the rods and cones (and thus serves as
  the "film"
• of the eye). The retina also has many
  interneurons that
• process the signals arising in the rods and cones
  before
• passing them back to the brain.

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Overview of the molecular basis for vision
The complex of 11-cis Retinal and a protein
called opsin forms the visual pigment, rhodopsin
(visual purple). Absorption of a photon of light
catalyzes the isomerization of 11-cis retinal to
all-trans retinal and results in its release.
This isomerization triggers a cascade of events,
leading to the generation of an electrical
signal to the optic nerve. The nerve impulse
generated by the optic nerve is conveyed to the
brain where it can be interpreted as vision.
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Recycling of retinal
11-cis Retinal is isomerized by light to
all-trans retinal, which converts to all-trans
retinol. All-trans retinol can be transported
across the interphotoreceptor matrix to the
retinal epithelial cell to complete the visual
cycle. Retinol is also transported to the retina
via the circulation, where it moves into retinal
pigment epithelial cells. There, retinol is
esterified to form a retinyl ester that can be
stored. When needed, retinyl esters are broken
apart (hydrolyzed) and isomerized to form 11-cis
retinol, which can be oxidized to form 11-cis
retinal.
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Structures of cis and trans of retinal
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The Receptors There are two classes of receptors
in the retina rods and cones. These receptors
are connected to the optic nerve. Cones are
individually connect to individual optic nerve
fibers. Multiple rods converge onto single
optic nerve fibers. It takes only one quantum
of light energy to activate a rod, but it took
several such hits for a threshold visual
response. It would seem therefore that rods
would have an advantage over cones because rods
can pool the signals by the convergence of
multiple receptors onto a single optic nerve
fiber.
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Detailed photocycle for vision
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• Rhodopsin has two components
• scotopsin, a protein moiety
• 11-cis-retinal, a carotene derivative.
• Energy from light excites the electrons in
  11-cis-retinal
• resulting in an isomerization in the excited
  state to form
• 11-trans-retinal. Because 11-trans-retinal is
  conformationally
• incompatible with the scotopsin moiety, it begins
  to detach,
• and the rhodopsin conjugate begins to break up
  into its
• component parts.
• The disintegration of rhodopsin into retinal and
  scotopsin is
• progressive, with a series of short-lived
  intermediates formed.
• Metarhodopsin II is an enzyme that ultimately
  effects the
• change in the rod membrane's charge. It acts to
  activate a
• second membrane-bound protein in the rod,
  transducin.

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Transducin is in its turn an enzyme activating
rod-resident phosphodiesterase, a third enzyme
in the cascade, capable of hydrolyzing cyclic
GMP. Cyclic GMP's role is to keep sodium
channels in the membrane of the rod open, so
that sodium flux is facilitated. In dark
conditions channels are open, sodium flux from
the extracellular space is approximately equal
to sodium loss via the pump system of the inner
rod segment, and the rod membrane is not
hyperpolarized. Under conditions of impinging
light, when the metarhodopsin
II--transducin--phosphodiesterase cascade is
initiated, cGMP is destroyed, sodium channels are
closed, and the flow of sodium ions into the rod
outer segment is slowed or stopped. This causes
it to become more negative, i.e., hyperpolarized
in the presence of light.
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Consider the rod in the diagram at left
membrane-bound pumps in the inner rod segment
(below the "waist") are actively pumping sodium
ions out. As fast as they are pumped out, the
outer rod segment brings them back in,
completing the circuit, provided the rod is
"dark." In the presence of light, however, the
transport of sodium back into the outer segment
is disrupted and the outer segment becomes
hyper- polarized. The interference with sodium
transport into the rod outer segment is mediated
by the cyclic decomposition and reconstitution
of the photoreceptor of the visual
pigment Rhodopsin.
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Four photopigments in human vision
There are four classes of photopigments as shown
in the above graph. The colors of the curves do
not represent the colors of the photopigments.
The colors of the curves do not represent the
colors of the photopigments. The wavelength of
maximum absorbance is indicated at the top of
each curve. The 420 curve is for the short
wavelength cones, the 498 curve is for the rods,
and the 534 and 564 curves are for the middle
and long wavelength sensitive cones
respectively.
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Spectral Sensitivity of Rods and Cones

• Bowmaker Dartnall (1980) projected a known
  amount of
• light directly through the outer segments of
  photoreceptors
• and measured how much light was absorbed by the
  photo-
• pigment molecules. There are three types of
  cones
• long-wavelength light (red)
• middle-wavelength light (green)
• short-wavelength light (blue)

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How can one pigment give different colors?
The spectrum of carotene is similar to that of
polyenes.
4 p electrons
6 p electrons
8 p electrons A
model carotene would have 10 p electrons.
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Particle-in-a-box model
A model cis-retinal has 8 p electrons.
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Particle-in-a-box model
A model all-trans retinal has 10 p electrons.
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Charge transfer states in polyenes
The charge transfer state shown below is a
resonance structure. The dipole moment for the
full charge separated state can be estimated
based on the length of the polyene. For the
decapentaene shown it is 9.82 Å. In the
presence of an applied electric field these
two states can mix. The environment of a protein
can provide such a field and can change the
optical properties of the molecule.
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Mixing of states in quantum mechanics
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Potential energy of a dipole in a field

• The interaction of an electric field with a
  dipole results
• in a change energy
• DU - mFcosq
• Consider two cases for the polyene shown.
• Aligned in a field of 107 V/m
• q 0o , cosq 1
• DU (47 D)(3.33 x 10-30 Cm/D)(107 V/m) 1.5 x
  10-21 J
• DU/hc 79 cm-1
• 2. Perpendicular to a field of 107 V/m
• q 90o , cosq 0, DU
  0

- - -

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The conversion of light into a nerve impulse
Hyperpolarization of a neuron in response to a
signal is rather unusual most neurons
depolarize, instead. But the rod does not, as
other neurons do, generate an action potential.
Nor does it release neurotransmitters. The
hyperpolarization response to the impingement of
light is proportional to light intensity and
thus the brighter the illumination the greater
the hyperpolarization. The net change in overall
membrane charge is perceived by the integrating
neurons of the retina, specifically the
horizontal and bipolar cells. They in turn pass
the information (with suitable inhibitory and/or
excitatory signals of their own) to the ganglion
cells. Ganglion cells, the last intra-ocular
neuronal element, send their axons out via the
optic nerves and into the visual processing
centers of the central nervous system.
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The conversion of light into a nerve impulse
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The conversion of light into a nerve impulse
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The conversion of light into a nerve impulse
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The conversion of light into a nerve impulse
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The conversion of light into a nerve impulse
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The conversion of light into a nerve impulse
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The conversion of light into a nerve impulse
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Hyperpolarization problem
Hyperpolarization occurs when the sodium flow
into the outer rod segment is stopped. This
causes a sodium gradient to build up. Assuming
that the sodium Concentration outside the cell
is 50 mM what concentration Must be reached
inside in order for the potential to be 50 mV at
37 oC?
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Hyperpolarization problem
Hyperpolarization occurs when the sodium flow
into the outer rod segment is stopped. This
causes a sodium gradient to build up. Assuming
that the sodium Concentration outside the cell
is 50 mM what concentration Must be reached
inside in order for the potential to be 50 mV at
37 oC?
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Phosphodiesterase kinetics problem
Phosphodiesterase (PDE) kinetics were determined
both In the presence and absence of an inhibitor,
rolipram. The apparent Km of PDE for cGMP was 5.3
1.0 µM with a competitive inhibition constant
for Ki 0.6 0.1 µM for the drug rolipram. The
maximum rate measured was Vmax 50
s-1. Determine the inhibitor concentration that
will reduce the Rate by a factor of 10 if the
cGMP 5.3 µM.
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Phosphodiesterase kinetics problem