Exam 1 Flashcards
(159 cards)
1
Q
Antoine van Leeuwenhoek
A
Built the first compound microscope to achieve significant magnification and observed unicellular organisms and plant tissues
2
Q
Antoine van Leeuwenhoek’s discoveries include:
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Single-celled organisms from pond water, red blood cells, spermatozoa
3
Q
Robert Hooke
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A contemporary of Leeuwenhoek who coined the term cell from looking at plant tissues
4
Q
Cell theory
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Formalized in 1838 by Matthias Schleiden and Theodor Schwann
- All living organisms are composed of one or more cells
- The cell is the most basic unit of life
- All cells arise from pre-existing living cells
5
Q
Camillo Golgi
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Discovered a way to stain a subpopulation of neurons using the golgi stain– this stain completely stains a neuron at random via an unknown mechanism
6
Q
What theory did Camillo Golgi propose?
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“Reticular theory”– the idea that neurons are continuous rather than physically separate cells
7
Q
Santiago Ramon y Cajal
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Described neurons as being discrete– contiguous, not continuous
8
Q
What is neuron doctrine?
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The idea that the nervous system is made up of discrete individual cells
9
Q
Theodor Meynert
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A Viennese psychiatrist who noticed regional variations in structure of different parts of the gray matter in cerebral hemispheres– tried to link psychiatry with histology and is considered the founder of cerebral cytoarchitecture
10
Q
How do we visualize neurons?
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We can dye them randomly (golgi) or individually (dye injection); we can also dye entire populations of cells based on macromolecule specific dyes like Nissl or Cresyl Violet
11
Q
What do Nissl and Cresyl Violet do?
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Bind nucleic acids
12
Q
What is the HRP enzyme?
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An enzyme that uses H2O2 to oxidize a substrate, which then yields a color change.
13
Q
What ways of classifying neurons are there?
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Shape, what emerges from cell body, number or branching of dendrites, branching of axons
14
Q
All neurons have ___ axon
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1 primary (primary meaning emerging directly from cell body)
15
Q
Bipolar neurons
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2 processes extending from cell body
16
Q
Pseudounipolar cells
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2 processes capable of generating action potentials; one extends to spinal cord, other towards skin or muscle
17
Q
Multipolar neurons
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many processes from cell body, but only 1 is an axon
18
Q
How do neurons differ from cells?
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Big with complex morphology and lots of surface area
Excitable– must maintain ion gradient
Energetically demanding
Post-mitotic (non-dividing)
Must signal and store info on short and long timescales
19
Q
Limit of resolution in Electron microscopy
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r=0.61lambda/NA (shorter wavelength= smaller r so higher resolution
20
Q
Electron microscopy provides _____ than visible light
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higher resolution (due to shorter wavelength of EM radiation)
21
Q
Transmission electron microscopy
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High-resolution images of thin slices of the object- electron beams pass through the object, creating 2-d image
22
Q
Scanning electron microscopy
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Slightly lower resolution images of topogrophy of thicker object- 3D image and electrons bounce off of subject
23
Q
Cytoskeleton types
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- Actin filaments
- Intermediate filaments
- Microtubules
24
Q
Actin filaments
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Aka microfilaments, are two-stranded helical polymers made from actin. They are flexible, 5-9 nm in diameter, organized in bundles, 2d networks, and 3d gels– they are usually found mainly in the cortex just beneath the plasma membrane
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Microtubules
Long, hollow cylinders made from tubulin, more rigid and 25 nm in diameter-- alpha and beta subunits arranged into a linear protofilament (12 in animals)
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Neurofilaments
10 nm in diameter and form the neuronal cytoskeleton along with microtubules and microfilaments (7 nm). Many monomers make coiled dimers, which join to make a terameric protofilament, making a protofibril which makes a filament
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Microtubules
Hollow tubes with 13 columns of tubulin molecules, 25 nm with a 15 nm lumen, made of a and b tubulin-- cell shape, motility, chromosome and orgamelle movement
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microfilaments (actin)
two strands of actin, 7 nm, cell shape, contraction, cytoplasmic streaming, motility and cleavage furrow
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intermediate filaments
neurofilaments-- proteins coiled into cables, 8-12 nm, usually keratin based, cell shape, anchoring nucleus and organelles, nuclear lamina
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Dendrites and axons have many
microtubules
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Are microtubules polarized?
Yes, they have directionally aligned polarity (alpha and beta ends) which allows for directionality
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Microtubule protein in axons
tau
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microtubule protein in dendrites
map2
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Cytoskeleton in neurons
the cytoskeleton provides routes for proteins, vesicles, and organelles to and from soma and distal regions of neuron
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Axonal transport is important in
presynaptic terminal function (microtubules)
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Steps in transport within neuron
1. synthesis, export
2. axonal transport
3. neurotransmitter release, membtane recycling
4. retrograde transport for degradation or reuse
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Actin in neurons
supports cell surface, supports smaller processes like dendritic spines
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Dendritic spine types
thin, stubby, mushroom
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Endoplasmic reticulum
free and membrane bound polysomes translate mrna, which is transcrbed from dna and emerges into cytoplasm to form polyribosomes (complex with ribosomes) -- secretory and membrane proteins translocate into rough er
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What does the cytoskeleton do?
provide routes for proteins, vesicles, and organelles to traffic to and from soma and distal neuronal regions
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What does active transport need?
atp motors walking along cytoskeleton
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Anterograde
away from soma
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retrograde
towards soma
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Which organelles are unique to the cell body in neurons?
Nucleus
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T or F: dendrites do not contain ER or golgi bodies
F
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T or F: protein synthesis can occur distally to the soma
T
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How does mRNA travel?
distally via the cytoskeleton
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Dendrites and their organells
ribosomes to translate mRNA (allows for local modification of protein structure and function), ER, and Golgi, allowing for transmembrane and secreted protein translation
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Role of ER in neuron
calcium source/buffer for signalling
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Where are mitochondria found in neurons?
distributed everywhere, especially synapses- they provide energy for areas of high metabolic demand
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Axons are myelinated by
oligodendrocytes (CNS) and schwann cells (PNS)-- provide a sheath that insulated axons and allows quick conduction of electrical impulses
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Astrocytes
make close contacts with synapses
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Electron microscope
uses electrons with a wavelength of >.3 angstroms, uses fixed (killed) samples to see dine details, organelles, vesicles
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light microscopy
uses visible light 350-700 nm on live or fixed samples, shows cell morphology, larger organelles, cell identity based on markers of proteins or nucleic acids
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transmitted light microscopy
uses white light, contrast derives from the interaction of light through the specimen (diffraction); low contrast, so optics enhance contrast
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how do cells affect visible light?
samples can lower the amplitude or make the lightwave go out of phase, or both
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phase microscopy
the sample separates out the phase shifted light from unaffected light, separating the signal from the specimen from everything else
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fluorescence microscopy
uses a specific wavelength of light for illumination, and collects specific lower-energy longer wavelength light. it relies on fluorophores (molecules with fluorescent priperties) and identifies cell morphology, organelles, cell types, and organelles
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Stokes shift
the shift from a higher energy absoption to the emission of a lower energy (longer wavelength)
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What are some advantages of fluorescence microscopy?
low background and high signal, making it easy to distinguish; it can also visualize specific cells, cell types, organelles, proteins/macromolecules, etc though macromolecules are at a lower resolution than light microscopy
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DAPI stain
a fluorescent dye that binds dna, helping visualize the nucleus
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mitotracker stain
fluorescent dye that accumulates in mitochondria
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Immunofluorescence
antibodies have a variable region that binds an antigen; adding a fluorophore to the antibody causes immunofluorescence; you can either use primary or secondary antibodies
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why might one use secondary instead of primary antibodies?
multiple secondary bind to a primary, creating signal amplification
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GFAP
red immunofluorescent dye for astrocytes
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map2
immunofluorescent blue or green dye for neurons
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uses of immunofluorescence
specific cells, organelles, subcdellular structures
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GM130 (green)
golgi apparatus
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PSD-95
postsynaptic density labelling (immunofluorescence)
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gfp
immunofluorescence to visualize neurons
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synapsin
immunofluorescence for presynaptic terminal
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if lots of psd-95 (red) and synapsin (blue/green) are present:
a synapse is likely there
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steps to fluorescent tagging
fusion gene of fluorescent protein and promoter, targeting sequence, and or protein of interest, and introduce gene into cells or organisms
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D1 dopamine receptor
drives td tomato expression
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d2 dopamine receptor
drives egfp expression
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advantages of fluorescent tagging
many colors, high brightness and specificity, helps see morphology and protein distribution, can use cell type promoters to drive fp expression, and fps can show enzyme activity, calcium dynamixs, protein interactions, etc
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cells of the nervous system
Neurons, glia (astrocytes, microglia, oligodendrocytes)
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astrocytes
metabolic and other support
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microglia
immune cells
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oligodendrocytes/schwann cells
myelination in cns/pns- insulate axon to speed up electrical conduction of impulses with regular breaks called nodes of ranvier; myelin extends from oligodendrocyte and weaps around multiple times
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do regular immune cells enter brain
no so microglia scavenge brain for debris
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Iba1 immunostaining
a specific microglia protein that stains microglia
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astrocytes
structural, metabolic, and trophic support for neurons, and helps form the blood brain barrier; also plays roles in plasticity and other processes. for example, astrocytes clear extra glutamate from the synapse
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how is cell membrane potential reached
separation of net charges across membtane (more positive ions outside)
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how do we measure cell potential
insert microelectrode into nerve cell, keep extracellular reference electrode- resting potential is about -60 mn
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why is there an electrical potential across the plasma membrane?
the plasma membrane is selectively permeable to ions, and a concentration gradient is actively established across the plasma membrane by pumps; there is also an electrochemical gradient as ions run down concentration gradient and electrical potential
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Na/K+ pump
uses atp to pump ions against concentration gradient, pumping Na out and allowing K in
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Ion channels
passively allow ions to pass through membrane and are selectively permeable (opened/closed easily)
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Resting Na/K gradient
Na tries to enter cell, K+ tries to exit but some are pulled back by the intracellular negative anions; cell is more permeable to sodium
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Potassium, Na, Cl, and Ca inside vs outside the cell
more inside, more outside, more outside, more outside
there are more organic anions inside the cell
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what is the simplified nernst equation
E(mV)= 58 log (ion outside/ion inside)
add a negative sign or flip when dealing with chlorine
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How could a neuron make its membrane potential less negative?
Allowing sodium or calcium into the cell can cause depolarization
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How can a neuron make its membrane potential more negative?
this is hyperpolarization; allowing K+ out or Cl- into the cell would allow this
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Graph of membrane potential vs log [K out/Kin]
a positive slope of 58 mV for 10fold increase in K+ gradient
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neurons have two types of changes in membrane potential:
graded depolarization (makes cell more excitable/positive)
action potential
graded hyperpolarization (makes inside more negative)
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are action potentials all or none
yes; graded potentials do not always cause APs
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threshold for an AP
-50 mV
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graded or passive action potentials
occur in dendrites, soma, axon w/ different duration and amplitude; spreads from origin in all directions and reflects local change in membrane permeability, decays with distance
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action potential
occurs only in axon with a defined duration and amplitude, travels away from soma, requires sequential change of membrane permeability ('travels' down axon)
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does the nernst equation account for permeability?
no, but when compared to measured membrane potential may tell us about membrane permeability
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Goldman equation
predicts Vm when membrane is permeable to multiple ions (Cl is negative so we flip it to in/out)
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Goldman equation
Vm= 58mV log (PKout+PNaout+Pcl in/ PKin+PNain + P cl out)
usually membrane so impermeable to Cl that you drop it
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earlier hypothesis of action potential
action potential is when membrane potential briefly goes to 0
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hodgkin and huxley
measured membrane potential during an action potential
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voltage clamp
used to measure ion currents in squid giant axons, hold voltage constant by injecting current, predicted ap changes from nernst and channel permeabilities
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describe the setup of the voltage clamp
one internal electrode measures membrane potential and connects to the voltage clamp amplifier, which compares potential with the desired potential; if its different, it injects current through a second electrode, this current flowing back into the cell is measured
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what did the voltage clamp experiment show
peak of ap is more positive than expected
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current when hyperpolarization occured
capacitive current but nothing else
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current when depolarization occured
capacitive current, transient inward, delayed outward
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how does voltage clamp work with depolarization
the membrane is depolarized and membrane potential is held constant, so that the currents undelying the ap are triggered but an ap cannot happen because potential is constant
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what happens to voltage clamp if no extracellular sodium
early current is outward instead of inward
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what happens if k+ current is blocked?
inward current but no outward current
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what did hodgkin and huxley find
29 mv is peak of ap, cell is selectively more permeable to Na than K
an early influx of sodium followed by delayed efflux of K+
using this permeability info we can calculate Vm
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how does depolarization affect conductance
increases conductance of sodium and potassium
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na vs k currents
at threshold, na and k are activated; na is fast and transient, k is slower and longer-- quick depolarization to E na followed by return to Ek
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how does the action potential travel down the axon?
there is a passive spread of charge in membrane potential--- the change in membrane potential decreases with distance, and current injected follows the path of least resistance to the extracellular space
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what is the change in vM mathematically
decays exponentially with distance from the site of current injection
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what does lambda length constant mean
the distance at which Vm is 37% of its value at the point of current injection
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describe the propogaction of an acrtion potential
an AP going from right to left causes a difference in potential in 2 adjacent regions; this difference creates a current that facilitates the passive spread of current so current spreads ahead and behind the AP
however, since more K exits the cell following the AP, the previous area is hyperpolarized and the sodium coming in isn't enough to depolarize
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why does the action potential travel only one way down the axon?
depolarization goes in both directions but the inactivation of VGNa+C prevents depolarization from reactivating the AP
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closed vs inactive voltage Na
in the resting state, the activation gate is closed and inactivation gate is opened
when activated Na flows in
then the inactivation gate closes and after repolarization the inactivation gate is open but the activation gate is closed
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what does it mean when they say propagation of an action potential is both passive and active?
passive because local current flow spreads to adjacent areas, causing depolarization
active because this causes the opening of voltage gated channels- the action potential mechanism
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nodes of ranvier
nodes in a myelinated axon that facilitate saltatory conduction
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what does myelin do
increase action potential conduction speed
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squid giant axon
no myelin, 25 m/s, 500 um
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motor axons
a alpha and a gamma, both myelinated
80-120 m/s, 13-20 um
4-24 m/s, 5-8 um
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sensory axons
alpha, beta, delta all myelinated (delta is thin), c is unmyelinated
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autonomic nerves
preganglionic b type is myelinated, postganglionic c type is not
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the majority of macromolecules in the cell are
proteins
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carbohydrates
energy storage, protein modification
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lipids
membrane and energy storage
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nucleic acids
dna and rna
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proteins
everything else
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how are amino acids added on
to the c terminus, the n terminus is 1
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protein shorthand
1 letter code, then the nth amino acid in a protein
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proteins are joined by
polypeptide bonds
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protein structure levels
1- amino acid sequence
2- substructures like alpha helices and beta sheets
3- full 3d structure
4- multiple proteins
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monomeric
no quarternary structure
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monomer
1 protein, may or may not participate in 4 structure
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multimeric
a protein complex with 4 structure
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subunit
1 protein in a multimer
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both pumps and ion channels are
multimers
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describe the process of x-ray crystallography
purified, concentrated protein solution yields crystals of the protein; an x-ray beam is shot through to get a diffraction pattern of electron density since the beam is scattered and scattered waves reinforce eachother now and then
this diffraction pattern helps produce an atomic model along with the sequence
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what do molecular pumps do
set membrane potential, transport ions and molecules directionally, and require energy from ATP or another gradient
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describe the structure of the na k pump
it has domains for nucleotide binding, phosphorylation, an actuator domain that acts as a dephosphorylase
adp binds to the nb domain and 2 k in, 3 na out
atp phosphorylates the pump which causes the na pump to turn outward and decrease affinity for na, after which a k binds , phosphate is released and pump goes to original conformation
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structure of the serca Ca2+ pump
areas for nucleotide binding, phosphorylation, ion translocation activity
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what does the ca pump do
2 ca out, 2-3 h in
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ATPase pumps
hydrolyze atp into adp, pump ion across membrane gradient
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domain of sodium potassium pump
ouabain inhibits na k pump
3 na ions bind
2 k ions bind
ATPase domain
pump itself gets phosphorylated
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alpha subunit of Na/K pump
alpha subunit has phophorylation site, atp site, ouabain site, and na/k site
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beta subunit of Na/K pump
helps with maturation and structure
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what changes are associated with the conformational change of the na k pump
changes in location pf phosphorylation, nucleotide binding, and activator domains
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pattern of na efflux
na efflux is reduced when extracellular k is removed, k+ restores recovery, efflux is again inhibited by metabolic inhibitors like dinitrophenol which blocks atp synthesis, recovery when atp is restored
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what role does the na k pump play in setting vm
sets up gradient for resting membrane potential and action potential-- but ONLY -1 MV
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how much do pumps and leak channels contribute to resting membrane potential
K+ leak current provides about -64 mv to resting membrane potential
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ion exchangers
use energy from existing gradient to pump another ion/molecule AGAINST its gradient (antiport since in different directions)
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co transporters
use energy from an existing gradeint to pump another ion against its gradeint but in the SAME DIRECTION
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