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276 Cards in this Set
- Front
- Back
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What is neurobiology the study of? |
- The study of nerve cells (neurons) and how they are organized to form the nervous system which mediates behavior
- The study of the way the system develops |
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What is the main function of the nervous system?
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- Signaling and information transfer
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Very basically, how does the nervous system send signals?
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Electrically, with ions - changes in membrane potential (voltage across the membrane)
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What does it mean to call the mechanisms of neuron signaling "basic"?
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Found universally throughout the animal world; do an essential job
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What are the three types of ways membrane proteins can be activated?
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- Voltage-gated (certain membrane potential)
- Ligand-gated (binding of specific chemical) - Mechanical deformation (stretch) |
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Based on the number of neurons, how are large systems different from small systems? Examples?
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Large systems = 10^10 neurons (i.e. human brain)
Small systems = 10^3 - 10^5 neurons (i.e. invertebrates or small ganglia) |
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What is an "identified neuron"? What type of system is it found in?
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- Neuron which is identified by its pattern of synaptic connectivity to other cells (muscles or neurons)
- It can be found reproducibly in other individuals - In small systems |
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How do "identified neurons" correspond to large systems?
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Large systems have individual neurons multiplied, so that there are types of neurons instead of individual neurons
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Who discovered the stain which brought a breakthrough into the study of neurons? What did the stain do?
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- Camillo Golgi
- Stained only a fraction of the neurons in the system, but stained them in their entirety |
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Who used the Golgi method extensively?
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Santiago Ramon y Cajal
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What was the disagreement between Golgi and Cajal about?
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- Golgi believed his stain did not stain each cell completely; he thought that neuron cytoplasm was interconnected in a continuous network
- Cajal believed the stain stained the neurons completely and that they were discrete cells; he was correct |
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What was the reticular theory and who came up with it?
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Camillo Golgi - believed that the stain of each cell was incomplete and that the cytoplasm of all neurons was interconnected to form a continuous network
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How was Cajal's theory, that neurons were discrete units that were completely stained by the Golgi stain, confirmed?
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- Electron microscopy
- New method: microelectrodes inject dyes, enzymes or other markers into identified cells |
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What type of cells are neurons?
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Modified epithelial cells
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What's the difference between convergent and divergent neurons? Examples?
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- Convergent neurons - gather a lot of inputs and have relatively few outputs; elaborate dendritic fields (ex: purkinje cells in cerebellum)
- Divergent neurons - gather small numbers of inputs but distribute widely; simple dendritic fields and numerous synapses (ex: granule cells of cerebellum |
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What are the characteristics of the cell body? What structures are found in it?
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- Does not divide
- Not myelinated - Nissl substance (ER) - Microtubules - Neurofilaments - Very few synaptic vesicles |
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What are the characteristics of the dendrites? What structures are found in it?
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- Receives synapses; spines
- Not myelinated (except in usual circumstances) - Nissl substance (ER) - Many microtubules - Some neurofilaments - Very few synaptic vesicles |
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What are the characteristics of the initial segment? What structures are found in it?
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- Very beginning of the axon (right after the axon hillock-curvature)
- Not myelinated - A little bit of Nissl substance (ER) - Bundles of microtubules - Neurofilaments - Very few synaptic vesicles |
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What are the characteristics of the axon? What structures are found in it?
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- Smooth, does not receive synapses
- Often myelinated - No nissl substance (ER) - Some microtubules - Many neurofilaments - Very few synaptic vesicles |
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What are the characteristics of the terminals? What structures are found in it?
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- Make synapses
- Not myelinated - No nissl substance (ER) - No microtubules - No neurofilaments - Many synaptic vesicles |
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What neuronal regions can be myelinated?
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Axon primarily; dendrites in unusual cases
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What parts of the neuron contain the Nissl substance (ER)?
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- Cell body
- Dendrites - A bit in the initial segment |
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What parts of the neuron contain microtubules?
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- Cell body
- Dendrites - Initial segment - A bit in the axon |
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What parts of the neuron contain neurofilaments?
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- Cell body
- Dendrites (some) - Initial segment - Axon |
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Where are the synaptic vesicles?
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- Primarily in the terminals
- Very few found in cell body, dendrites, initial segment, and axon |
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What is the potential difference of a nerve cell membrane usually? How high would it have to be to destroy the membrane?
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Usually: -50 to -85 mV
Destroy: -110 mV |
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How does the excitatory and inhibitory response differ?
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- Excitatory - depolarizes (makes potential more positive)
- Inhibitory - hyperpolarizes (makes potential more negative) |
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What are nuclei or ganglia?
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Groups that cluster together cells with similar functions
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Are there ribosomes in the axon?
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Yes, although the density of ribosomes is very low relative to the cell body and dendrites
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Can protein synthesis occur in the axon?
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Yes, because there are ribosomes there (although very few), especially in regenerating axons
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What are the four different speeds at which components can be transported in the neuron?
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- Fast (240 mm/day)
- Intermediate (30-60 mm/day) - Slow (2-8 mm/day) - Very Slow (1 mm/day) |
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What is carried at the fast speed (240 mm/day) through the neuron?
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Lipids, plasma membrane proteins (Na+ pump), and synaptic vesicles
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What is carried at the intermediate speed (30-60 mm/day) through the neuron?
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Mitochondria
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What is carried at the slow speed (2-8 mm/day) through the neuron?
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Bulk cytoplasm, e.g. actin and metabolic enzymes
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What is carried at the very slow speed (1 mm/day) through the neuron?
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Cytoskeletal proteins (microtubules and neurofilaments)
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What does fast and intermediate transport utilize?
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Microtubules (note these are transported at a very slow speed, but are used during fast and intermediate transport!)
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What two proteins are involved in movement along microtubules?
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- Kinesin (orthograde = forward)
- Dynein (retrograde = backward) |
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What does kinesin use for orthograde transport?
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- Binds to synaptic vesicles via an adaptor protein
- "Walks" along the microtubules with energy from ATP |
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Where are microtubules polarized? Which way is which?
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Axons: + end towards synaptic boutons; - end towards dendrites
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What happens at the positive end of microtubules?
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There is a lot of turnover of tubulin at the + end
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Which way down the microtubules does kinesin move? Dynein?
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Kinesin moves towards the + end
Dynein moves towards the - end |
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What does kinesin carry? What does dynein carry?
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Kinesin: synaptic vesicles
Dynein: endosomes, Golgi apparatus, ribosomes |
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How can the polarity of microtubules be determined?
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- Inject excess tubulin
- Tubulin will add to the sides of tubules forming sheets that are curved - Fix cell, section it, and examine with electron microscopy - Observe hooks curving: clockwise + end coming towards you; counterclockwise - end is coming out at you |
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When excess tubulin is injected into cells to determine the direction of polarity, what does the clockwise and counterclockwise refer to and mean?
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CW and CCW refers to the direction the tubulin hooks are curving
- CW = + end coming out - CCW = - end coming out |
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Where are the microtubules all aligned? How so?
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In the axon, microtubules are aligned with the same polarity; + towards the end of the axon; - towards the cell body
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Where are microtubules not aligned?
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In the dendrites microtubules are arranged with both polarities
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Why is it suspected that ribosomes are not frequently found in axons?
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Rough ER and polyribosomes stick to dynein so if they were to go down the axon they would be brought rapidly back to the cell body
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What molecules use adaptor proteins?
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Dynein and Kinesin (in order to transport their respective cargos down the microtubules)
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When excess tubulin is injected into cells to determine the direction of polarity, what does the clockwise and counterclockwise refer to and mean?
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CW and CCW refers to the direction the tubulin hooks are curving
- CW = + end coming out - CCW = - end coming out |
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Where are the microtubules all aligned? How so?
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In the axon, microtubules are aligned with the same polarity; + towards the end of the axon; - towards the cell body
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Where are microtubules not aligned?
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In the dendrites microtubules are arranged with both polarities
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Why is it suspected that ribosomes are not frequently found in axons?
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Rough ER and polyribosomes stick to dynein so if they were to go down the axon they would be brought rapidly back to the cell body
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What molecules use adaptor proteins?
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Dynein and Kinesin (in order to transport their respective cargos down the microtubules)
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What are the glial cells called in the PNS and CNS respectively?
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PNS: Schwann cells
CNS: Oligodendrocytes, Astrocytes, Microglia |
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What is the function of Schwann cells?
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They envelop axons, either simply as in unmyelinated axons, or more elaborately by wrapping a myelin sheath around the axon
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How much of an axon does each Schwann cell cover?
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Each Schwann cell is responsible for only a short length of axon
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What is the gap between myelin segments called?
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Nodes of Ranvier
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What is the ratio of glial cells to neurons?
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10-50x as many glial cells as neurons
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What is the function of oligodendrocytes?
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Make central myelin; they can enwrap several axons
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How many axons can a Schwann cell myelinate?
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One
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What is the structure of oligodendrocytes?
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Branched cells and the end of each branch differentiates into a spade-like expansion that does the wrapping
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What do astrocytes do?
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They use their end feet and apply them to the surface of neurons and cerebral blood vessels
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What are microglia do?
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Phagocytic cells; clear up the mess after a neuron dies
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What are the functions of glial cells?
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- Removal (by active transport across the membrane)
- Metabolism of neurotransmitters present in the extracellular space - Buffering of external ion concentrations - Guidance of neuronal migration during development - Establishing the blood-brain barrier possibly - Possible nutritive functions |
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What does the axon look like? Dendrites?
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Axon - long and cylindrical, usually not branched
Dendrites - start wider and get skinnier and branch |
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What is the function of the Golgi?
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Processes and packages protein vesicles to send down the axon to the terminal
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What is the function of the Nissl substance? What is it like?
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Takes part in protein synthesis; like the rough ER
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What are microtubules? Diameter?
What are neurofilaments? Diameter? |
Element of the cytoskeleton: 25nm
Intermediate filament: 10nm |
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What is the part of the neuron that gives rise to the initial segment where the action potential is initiated?
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Axon Hillock
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What type of axons are more likely to have protein synthesis / ribosomes?
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Regenerating / growing axons
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Does a larger stimuli or longer stimuli affect the action potential?
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Yes, but not by making it larger or longer (rather it fires more frequently)
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An action potential causes what change in voltage and lasts for how long?
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0.1V pulse
0.001 seconds (1 ms) |
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For an action potential, the inside of the cell has what charge? During an action potential it has what charge?
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Negative before
Positive during |
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How fast can an action potential move?
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120 m/s (270 mph)
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How are glial cells unique from neurons in terms of differentiation/regeneration?
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Glial cells can still divide in mature animals unlike the majority of neurons; this, however, is a common source of tumors
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How do glial cells differ from neurons structurally?
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Glial cells do not have axons
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How are glial cells connected; how do they communicate with each other?
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Gap junctions
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What can pass through the blood-brain barrier? What can't?
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Can: lipophilic molecules (such as alcohol) and gases
Can't: proteins, ions, hydrophilic molecules |
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What is the most common way to record electrical signals?
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Penetrate the nerve cell with a microelectrode
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What is the purpose of using a microelectrode?
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It allows you to measure the voltage between the inside and outside of the cell
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How is a microelectrode constructed?
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A glass capillary is drawn out to a fine tip and filled with a salt solution (conducts electricity); tip diameters are 0.1-0.5 μm
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Why is glass a good material to make microelectrodes from?
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It sticks to the cell membranes creating a tight seal - preventing the hole it makes from acting as a short circuit
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What method confirms that glass capillary microelectrodes are measuring the membrane potential accurately?
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Certain fluorescent dyes absorb onto cell membranes and the intensity of their fluorescence is proportional to the membrane potential.
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Glass microelectrodes used in intracellular recording have resistances of what value?
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10^7 - 10^8 ohms
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The axon is responsible for propagation of action potentials and uses what kind of channels?
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Voltage-Gated (Na+, K+) (Ca+ in presynaptic terminal)
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The dendrites receive synapses and use what kind of channels?
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Ligand-Gated (neurotransmitters)
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Where can synapses be received?
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Dendritic spines (usually) but can also just be on the dendrite
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Which type of glial cell looks like a star? What is it known for?
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Astrocytes - send out messages in many directions; communicates with epithelial cells of blood vessels and neurons
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Oligodendrocytes are in which nervous system and do what?
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CNS - can make more than one myelin sheath around separate axons (however multiple oligos / axon)
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Which glial cells are involved in the blood-brain barrier?
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Astrocytes because they make tight junctions
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What are the different recording techniques?
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- Glass electrodes (sharp - penetrates membrane) (patch - grabs piece)
- Wire electrodes |
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If you have a very thin glass capillary measuring the membrane potential, what will happen when you stick another microelectrode into the cell?
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There will be a slight blip in voltage but it will return to the same voltage (-70mV) showing that you didn't injure the cell
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What is the glass microelectrode filled with?
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3M KCl
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What machine is used to record the voltage? What is its resistance?
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Oscilloscope
10^6 ohms |
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Why must a preamplifier be used?
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It has a larger resistance than the microelectrode (10^7-10^8) with a resistance of 10^10 - 10^12 ohms. This causes the largest voltage drop to be across the pre-amplifier; the oscilloscope which is attached in parallel will then more accurately measure the voltage.
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What happens if a preamplifier is not used with the microelectrode and oscilloscope?
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The greatest voltage drop will occur across the electrode and only 10% will drop across the oscilloscope which will not give an accurate depiction of the voltage.
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How much larger must the resistance across the pre-amplifier be compared to the microelectrode?
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100x higher
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When are wire electrodes used?
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To measure the electric activity extracellularly; they are useful for measuring action potentials in single cells of the brain
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What can and can't wire electrodes do?
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They can't measure membrane potentials but they do detect the currents set up in extracellular fluid by currents flowing across the membranes due to changes in membrane potential
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Do wire electrodes (extracellular recordings) have higher or lower amplitudes compared to intracellular recordings?
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Wire electrodes are lower in amplitude
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What are the three types of brain imaging techniques?
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1. EEGs (electroencephalograms)
2. PET (positron emission tomography) 3. fMRI (functional magnetic resonance imaging) |
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How is an EEG done?
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Made by recording electrodes placed on the scalp; they record gross activity in general areas of the brain
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How does PET work?
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Uses positron-emitting isotopes to follow the localization of specific molecules in the brain; for example 15-O glucose is taken up into cells that are active during particular behaviors/tasks so you can map the region of the brain that is active
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What technique is used to detect the localization of certain neurotransmitter molecules?
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PET
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What is the resolution of PET? of fMRI? of MRI?
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4mm
3mm 1mm |
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How does an fMRI work?
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Detects the difference in magnetic moment of oxy and deoxy hemoglobin, which correlates to active regions which use more oxygen
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Why is the fMRI advantageous over the PET scan?
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It does not involve radioactive isotopes
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The knee-jerk reaction is characterized by what?
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- Automatic
- Simple neural circuit - Monosynaptic reflex |
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How does the knee-jerk reaction occur?
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- A stretch receptor in the quadricep muscle is activated which generates a sensory-receptor potential
- If large enough it becomes an action potential and travels through the dorsal root to synapse at the spinal motor neuron - This causes a synaptic potential at the motor neuron which sends an AP to the muscle where it twitches |
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How is long distance signaling transmitted?
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Action potentials
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How are signals transmitted across synapses?
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Synaptic potentials
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What does integration refer to?
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Decision-making; whether to fire an action potential or not
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What are the three types of electrical signals?
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1. Receptor potentials
2. Action potentials 3. Synaptic potentials |
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A negative current generates what? A positive current generates what?
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Negative => Hyperpolarization
Positive => Depolarization |
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What is necessary to generate an action potential?
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A positive current which depolarizes the membrane enough to pass a certain threshold
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Which animal was important for studying the action potential?
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Squid giant axon (d=0.5mm)
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Why is the squid axon so large?
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Most invertebrates don't have myelin so increasing the conduction velocity is achieved by increasing diameter
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What is the resting potential of an axon?
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-70 mV
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How is a current measured and generated in an axon artificially?
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- A microelectrode is inserted into the axon
- A second microelectrode is inserted and used to pass current to change the potential |
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Once the threshold for an Action Potential is exceed it is called?
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All-or-Nothing
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How long is an action potential?
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2 msec
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What animal is used to learn about receptor potentials?
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Crustaceans/crayfish - the stretch receptor
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Why are crustaceans/crayfish used for studying receptor potentials?
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The stretch receptor neuron has a large cell body, located right next to the stretch detector part of the cell; the cell body makes for a nice target for microelectrodes near the action
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How are receptor potentials and synaptic potentials different from action potentials?
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- Local (does not propagate)
- Graded with stimulus strength (vs. constant amplitude) - No threshold - Not all-or-nothing - No refractory period |
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How does a receptor potential occur?
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When stimulus is applied, the receptor depolarizes with an amplitude that increases as stimulus increases (graded and no threshold)
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When the receptor potential reaches a certain threshold what occurs?
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An action potential is generated
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The amplitude of a receptor potential is communicated how with action potentials?
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The frequency of AP's
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What is the timing of a synaptic potential relative to an AP?
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10msec vs 2msec
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If one pathway is stimulated more than once within the 10msec duration of a synaptic potential what happens?
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The amplitude of the second response is larger than the first (the two signals add in temporal summation)
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If more than one pathway is stimulated with a lag of less than the 10msec duration of a synaptic potential, what happens?
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The responses can also add together is spatial summation
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What are the two broad classes of synapses?
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Excitatory (E)
Inhibitory (I) |
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How is the simultaneous input of excitation and inhibition understood for synaptic potentials?
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They are summed with integration
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What in the membrane is responsible for the capacitance? For the resistance?
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Lipids = capacitance
Proteins = resistance |
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What is the purpose of cell membranes?
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Create and maintain the differences between the inside and outside of the cell; ion gradients
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How are ion gradients generated? How are signals generated?
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- Ion pumps generate ion gradients
- Ion channels use the ion gradients to generate signals |
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What is the ratio of lipid to protein in the membrane? (by mass, by moles)
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50:50 by mass
50:1 by moles |
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How thick is the membrane?
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5nm
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How is the membrane held together?
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Mainly through non-covalent interactions
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What does it mean to be amphipathic? What is an example of this?
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Containing both hydrophilic and hydrophobic parts (phospholipids, cholesterol)
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What are the four major phospholipids?
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- 3 with a glycerol backbone (phosphatidyl-choline, -serine, -ethanolamine)
- Sphingomyelin |
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What is found on the three hydroxyls of glycerol (for phospholipids)?
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- Two fatty acids, one is saturated and one is unsaturated, usually
- A phosphate with another molecule (choline, serine, ethanolamine, or inositol) attached |
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How are the head groups attached to the phosphates on glycerol?
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With a phosphodiester bond
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Phosphatidylinositol is important for what?
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Signaling pathways
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What gives side chains kinks? What does this cause?
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Unsaturation of fatty acids - decreases the ability of side chains to pack together so it increases the fluidity of the membrane
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Phospholipids in water tend to form what?
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Micelles
Bilayers |
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How can lipids in the membrane move?
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Laterally (pretty fast, 2 micrometers/second)
Spin about their long axis very fast |
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What move can lipids rarely do in the membrane?
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Flip from one monolayer to the other
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How does cholesterol orient itself in the membrane? How does cholesterol affect the membrane?
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Its -OH positions itself near the polar head groups; the hydrophobic part inserts between the fatty acid side chains leading to a stiffening of the membrane
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How are the membrane lipids asymmetrically distributed?
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Outside: choline-containing lipids (lecithin and sphingomyelin)
Inside: phosphatidyl-serine, -ethanolamine, and -inositol |
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How can artificial membranes be assembled?
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- Take a solution of lipid in chloroform and paint a drop of the solution over a hole in a Teflon barrier that separates two chambers containing water and aqueous solutions
- Chloroform is appreciably soluble in water so it slowly leaves the drop and concentrates the lipids; eventually the chloroform is gone and there is a bilayer of phospholipid left in the aperture |
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What is Ohm's Law?
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V = I*R
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In order to measure the currents carried by ions, what needs to be done?
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The ionic currents need to flow seamlessly into currents carried by electrons
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What is the common interface between ionic currents and electron currents?
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Silver/Silver Chloride electrode which consists of silver metal (wire or foil) with silver chloride deposited on the surface
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What happens at the anode of a Ag/AgCl electrode?
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Anode: Ag + Cl- ==> Ag+ + Cl- + e-
(AgCl is insoluble and gets deposited on the electrode; a chloride ion goes from solution onto the electrode) |
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What happens at the cathode of a Ag/AgCl electrode?
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Cathode: Ag+Cl- + e- ==> Ag + Cl-
(a silver ion is converted to silver metal in the electrode, and the associated chloride ion gets released into solution) |
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What is an amp?
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Measure of current: 1 coulomb / second
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What is the charge on a singly charged ion?
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1.602x10^-19 coulombs
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What is a Faraday?
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The charge (in coulombs) of one mole of univalent ion;
10^5 coulombs / mole |
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The resistance is calculated how?
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R = rho* L/A
(rho is the specific resistance, units ohm*cm) |
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Because in membranes it is hard to have an accurate measure of L, the thickness of the membrane, the resistive properties of membrane matter are described as:
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Rm = rho*L (units = ohm*cm^2)
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What is the resistance of an artificial membrane (units = ohm*cm^2)
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10^8 ohm*cm^2
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What is the resistance of a natural membrane (units = ohm*cm^2)?
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10^3 - 10^4 ohm*cm^2
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Are the resistive properties of a natural and artificial membrane similar?
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No, they are very different, because natural membranes have specific proteins in them that govern the resistive properties
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How many carbons are the fatty acids on phospholipids usually?
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12-24 (even numbers only)
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What is the structure of the head group Choline?
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(CH3)3-N+CH2-CH2-OH
(OH makes phosphodiester bond) |
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What is the structure of the head group Ethanolamine?
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+NH3-CH2-CH2-OH
(OH makes phosphodiester bond) |
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What is the structure of the head group Serine?
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(+NH3)(COO-)-CH-CH2-OH
(OH makes phosphodiester bond) |
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What is choline called when it is attached to a phospholipid?
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Phosphatidylcholine (lecithin)
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What enzyme can remove one of the fatty acid tails on a phospholipid?
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Phospholipase
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When a phospholipid only has one fatty acid tail, due to phospholipase, what is it likely to form?
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Micelles
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How does cholesterol affect membranes?
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It stiffens the outer part which decreases the fluidity
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What is a capacitor? How does this relate to the plasma membrane?
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2 conductors (solutions on either side) separated by an insulator (membrane)
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What is the equation for capacitors?
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Q=V*C
(Q = charge, in coulombs) (V = potential, in volts) (C = capacitance, in farads) Qm = V*Cm (Cm = farads / cm^2) (Qm = coulombs / cm^2) |
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If we apply a voltage across a capacitor, what will happen?
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A current will flow as charge accumulates
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What is current?
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The change in charge per change in time:
I = dQ/dt |
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What is the capacitance of an artificial membrane? of a natural membrane?
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1 μF / cm^2 = 1x10^-6 F / cm^2
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What is the capacitance due to?
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The lipids in the membrane
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How much charge is separated by the capacitance at the normal resting potential? How does this affect the cell?
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A tiny amount; the concentration in the bulk solution is not detectably changed by this separation of charge
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Why does the artificial membrane have a much higher resistance than the natural membrane?
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The natural membrane has proteins which acts as resistors in parallel (decreasing the overall resistance of the membrane)
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What are ionophores?
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Small hydrophilic peptide antibiotics made by bacteria or molds that make the membranes permeable to specific ions
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Why do ionophores exist?
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They are presumably to wage biological warfare against competitors or prey - they make the membranes of the organisms they attack permeable so that their ion gradients run down and die (bacteria and molds)
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What ionophore does Streptomyces fulvissimus make? What does it allow through?
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Valinomycin - doughnut-shaped molecule with a hole of just the right size and chemistry for K+
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How does valinomycin (from Streptomyces fulvissimus) function?
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The outside surface is hydrophobic so it dissolves in membranes; it shuttles K+ ions across the membrane through its doughnut-shape; leads to death
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What is the structure and function of gramicidin, an ionophore?
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- It is a molecule folded as a loose helix with a hole down the middle; two molecules form a pore in the membrane
- It is selectively permeable to K+ so it leaks out, flowing down the concentration gradient, leading to death |
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When gramicidin is added to an artificial membrane, what happens?
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The resistance goes to 1000 ohm-cm^2 (closer to the real membrane resistance)
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In the experiment with an artificial membrane and gramicidin, what was put on either side of the membrane? Why?
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1) 0.1 M KCl
2) 0.01 M KCL and 0.18 M sucrose - Sucrose was added so there won't be osmotic pressure differences - Gramicidin makes membrane permeable to K+ which will flow from 1 -> 2 (leaving Cl- behind) - Eventually concentration force and electrical force will equilibriate |
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What is the resting potential in an artificial membrane that is permeable only to K+ (due to gramicidin)?
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-58mV
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How does the experiment with a artificial membrane/gramicidin and 0.1M KCl vs. 0.01M KCl equilibrate?
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- Gramicidin makes the membrane permeable to K+
- K+ from 0.1M side flows down the concentration gradient leaving Cl- behind - + charge builds up on 0.01 side while - charge builds up on 0.1 side - Leads to a voltage across the membrane - Pos. charge opposes flow of K+ - Current continues until concentration force is balanced by the electrical force |
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What is the equilibrium potential?
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The membrane potential at which the net flow of K+ ions is zero (due to the electrical force)
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Once at the equilibrium potential, does K+ still move?
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Yes, the rate of flow with the concentration gradient exactly balances the rate of flow with the voltage.
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What is the Nernst equation used for?
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Tells the equilibrium potential with various concentrations of an ion
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What is the Nernst equation?
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Ek = (RT/F) ln [Ko]/[Ki]
Ek = 58 x log [Ko]/[Ki] @ 20 deg. C, in mV |
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Although natural membranes are permeable to more than just K+ ions, the resting potential (Vm) is close to what?
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The equilibrium potential for K+
(Ek) |
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If an external voltage is applied across a membrane that is in equilibrium, what happens?
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The membrane potential is shifted from Ek, K+ will no longer be at equilibrium and there will be a net flow of K+ (current) across the membrane
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When no current is flowing and the membrane is in equilibrium, Vm=?
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Vm = Ek
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How can the conductance (g) be measured
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Plot the current across the membrane (i) vs. the membrane voltage (Vm); the slope of the line gives us the conductance of the membrane (g)
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Conductance is the reciprocal of what?
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Resistance: gk = 1 / Rk (measured in Siemens = 1/ohms)
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When a voltage is applied to generate a current across a membrane, how can the voltage be determined?
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Vm = Ek +ik(Rk)
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What is the equation for current? (example using potassium)
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ik = gk(Vm - Ek)
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What does the current carried by an ion depend on?
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- The membrane conductance for that ion
- The electrical driving force that makes the current flow (the difference between the membrane potential and equilibrium potential for the ion) |
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What is the intersection of the line [that is plotted on the current (i) vs. membrane voltage (Vm)] with the voltage axis?
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Gives us Ek
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When ik=0, Ek =?
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Vm
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What are the units of specific resistance (Rm)?
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Rm = ohm*cm^2
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What equation does the exponential decay part of the voltage vs. time graph follow?
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V = Vo e^-t/T
T= time constant (determines shape) = RC (resistance*capacitance) |
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How are integral membranes attached?
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- Transmembrane
- Covalent linkages |
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What feature of transmembrane proteins makes them fit in the membrane?
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Amphipathic - hydrophilic surfaces exposed to outside and inside of cell; hydrophobic surfaces nestled in membrane
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Integral membrane proteins that are not transmembrane are mainly what (hydrophilic or hydrophobic)? How are they attached?
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Hydrophlic - attached via covalently attached fatty acids or by a GPI linkage
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What is the structure of the portion of the transmembrane protein that spans the membrane?
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20-25 amino acids (hydrophobic); alpha helical (forms about 6-7 turns to span membrane)
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How are the hydrophilic amino and carbonyl parts of the transmembrane protein taken care of in the hydrophobic lipid bilayer?
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They hydrogen bond with themselves leaving the hydrophobic side chains to stick outward where they can form hydrophobic interactions w/ the fatty acids
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What way are the hydrogen bonds of the alpha helix (in transmembrane proteins) oriented?
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Parallel to the long axis of the helix
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How can the transmembrane domains be predicted from the amino acid sequence?
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Hydrophobicity Plots (identifies 20-25 amino acids with hydrophobic side chains)
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Most transmembrane proteins have what modifications and where?
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- Glycosylated on the outside (non-cytoplasmic)
- Disulfide bonds are on the outside - Sulfhydryl groups on the inside of the cell (cytosol is reducing) |
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What are the two commonly used detergents?
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SDS (sodium dodecyl sulfate) and Triton X-100
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What is the purpose of detergents?
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Small amphipathic molecules that are needed to solubilize and fractionate membrane proteins in order to disrupt hydrophobic bonds
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How can proteins be fractionated after they are solubilized by a detergent?
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Using SDS-PAGE (polyacrylamid gel electrophoresis in the presence of SDS, sodium dodecyl sulfate)
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What is a "magic bullet" referring to?
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A toxin or drug that binds to the protein you want to isolate with very high affinity
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Besides alpha helices, what other transmembrane structures can go through?
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Beta-sheet channels arranged antiparallel for ideal H-bonding
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What is the electroplax? Who is it found in?
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In electric rays and eels, a special organ that generates electrical shocks, which is used by the fish for aggressive purposes
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How is the electroplax different in ray and the eel?
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In rays the electroplax has lots of ACh receptors while in the eel there are a lot of voltage sensitive Na+ channels
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What toxins bind to ACh receptors and which bind to Na+ channels?
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- ACh receptors - Bungarotoxin
- Na+ channels - Tetrodotoxin (TTX) |
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How can toxins such as Bungarotoxin (binds to ACh receptors) and Tetrodotoxin (binds to Na+ channels) be used to study proteins?
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These toxins can be radiolabeled and used to detect fractions enriched in the protein after fractionation procedures, and eventually lead to the isolation of pure protein; also can be used in affinity chromatography
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After you have purified a protein, what must you do?
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- Check whether it works and that it is the right protein - insert into artificial membrane
- Determine amino acid sequence, either directly or from cloned DNA |
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What are the three techniques to determine if the hydrophilic regions are inside or outside of the cell?
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1. Chemical Modification
2. Impermeant Proteases 3. Antibody Labeling |
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How does chemical modification work for determining which side of the membrane the hydrophilic regions of transmembrane proteins are on?
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- A labeled reagent that covalently bonds to certain amino acid side chains and which does not permeate the membrane is selected
- Intact cells are exposed to this reagent - Protein is isolated and cleaved into smaller peptides - Peptides are separated and those w/ label are identified and sequenced (these were on the outer surface) - Repeat with cells that are permeabilized to allow reagent internally (additional labels are internal hydrophilic regions) |
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How does impermeant proteases work for determining which side of the membrane the hydrophilic regions of transmembrane proteins are on?
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- Exposure to a protease that does not cross the membrane
- Identifies sensitive sites on the outside of the cell and after permeabilizing the cell, affects the inside too |
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How does antibody labeling work for determining which side of the membrane the hydrophilic regions of transmembrane proteins are on?
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- Small peptides can be synthesized, corresponding to the predicted hydrophilic domains
- Antibodies are raised against each peptide and then labeled w/ a marker - Each antibody then can be applied to the intact cell or to a permeabilized cell to distinguish the internal from external regions |
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What is the freeze-fracture technique?
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- Cell is flash-frozen w/ liquid nitrogen
- Cleaved in a vacuum with an axe; usually divides the two layers of the bilayer |
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After the Freeze-Fracture technique divides the two layers in half, what happens?
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Hot metal filament showers platinum atoms on the profile to "shadow" the membrane layer
Leaves behind a shadow which is observed under an electron microscope |
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When the freeze fracture technique is conducted, how the cell split?
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Fracture planes follow the path of least resistance; which in a frozen cell is the interior of the phospholipid bilayer (some proteins stay on either side)
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When the neurons at the neuromuscular junction are freeze fractured, what can be seen in the membranes?
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- The postsynaptic cell shows acetylcholine (ACh) receptor molecules
- The presynaptic cell shows Calcium channels arranged in double parallel lines |
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How can it be shown that the membrane is a fluid mosaic?
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- Label membrane proteins with antibodies in separate areas; with time (30 min.) they are mixed.
- FRAP |
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What does FRAP (fluorescence recovery after photobleaching) do?
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- Membrane proteins are fluorescently labeled
- A small area is photobleached by exposure to a bright spot of light - With time, fluorescence returns to the area as unbleached labeled proteins diffuse into the area |
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Some membrane proteins don't diffuse because of what?
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They are tethered, by attachment to the cytoskeleton
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What are some examples of tethered proteins?
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- Na+ channels at the nodes of Ranvier in myelinatend nerves
- Ca++ channels in the presynaptic membrane - Acetylcholine receptors in the postsynaptic membrane of the neuromuscular junction |
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What happens if you double the area of the membrane?
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- The capacitance will be doubled (twice as many charges can be stored)
- The resistance will be halved (there will be twice as many channels to carry the current) |
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If the membrane is permeable only to K+, then Vm =?
If the membrane is permeable only to Na+, then Vm =? |
Vm = Ek
Vm = Ena |
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In the squid giant axon, the ion concentrations of Na, K, Cl inside the cell are? (and impermeant anions)
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Na+ = 50mM
K+ = 500mM Cl- = 40mM Impermeant Anions = 385mM |
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In the squid giant axon, the ion concentrations of Na, K, Cl outside the cell are?
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Na+ = 440mM
K+ = 20mM Cl- = 560mM |
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In the squid giant axon, based on the concentrations of K+, Na+, and Cl- inside and outside the cell, what are Ek, Ena, and Ecl equal to?
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Ek = -75 mV
Ena = +55mV Ecl = -66mV |
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What is the resting Vm of a squid giant axon? How does this compare to Ek (supposed to be a good estimate)? What is this deviation attributed to?
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Vm = -65 mV
Ek = -75 mV There is a small permeability to Na+ at rest |
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How does the Nernst equation explain Vm depending upon the concentration of K+ outside of the cell?
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High Ko, Vm = Ek
Low Ko, Vm deviates from Ek |
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If the Ena = +55mV, what forces are causing the Na+ to flow? What direction?
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Both concentration force and the electrical driving force are making Na+ go through any open Na+ channels that are in the membrane (to enter the cell)
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Because the resting membrane potential is more positive than Ek, what will K+ ions do?
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K+ flows out of the cell because there is a net driving force since Vm is not at Ek
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When is the current of an ion zero?
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When it's E(k, na..) = Vm
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When is there a steady state?
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When there is no net current across the membrane
Ina + Ik (+ Icl) = 0 |
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If we only consider the flow of K+ and Na+ ions, which equation gives the resting potential, Vm?
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Vm = [(gna/gk) Ena + Ek] / [(gna/gk) + 1]
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What is the ratio of gna / gk in the squid gian axon?
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gna/gk = 1 / 10
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When the equation for Vm (focusing on the flow of only Na+ and K+) is used, what is the value for Vm?
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-63mV (very close to the measured value; which means that the deviation in the plot Vm vs log Ko can most be explained by Na+)
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- In some neurons Ecl is positive w/ respect to the resting membrane potential (RMP), what happens?
- What about when Ecl is negative with respect to RMP? |
- Positive: Cl- is pumped in
- Negative: Cl- is pumped out |
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When K+, Na+, and Cl- are taken into account, what is the equation for Vm?
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(gna*Ena + gk*Ek + gcl*Ecl) / (gna + gk + gcl)
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Does chlorine have an impact on the voltage?
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Hardly at all, we will ignore its role
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What is the resting potential due to?
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Largely due to a high K+ conductance, so largely influenced by Ek; but the resting potential is a bit more positive than Ek because there is a small permeability to Na+
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How do ionic gradients arise?
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Ionic pumps
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What does the sodium pump do?
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Uses the energy of ATP hydrolysis to pump out 3 Na+ ions and simultaneously pump in 2 K+ ions
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What happens if the sodium pump is poisoned?
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The ion gradients dissipate and the cell depolarizes
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What is the result of the sodium pump, pumping out 3 Na+ ions to every 2 K+pumped in?
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A net outward transfer of positive ions; a current; this contributes to the resting potential
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What contribution does the sodium pump's outward current do to the resting potential?
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About 2mV (changes it from -63mV, from Na+ and K+ permeability, to -65mv)
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How is the Cl- concentration in some neurons maintained?
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Action of the chloride-bicarbonate exchanger, which brings in 1 Na+ and one bicarbonate ion for each Cl- removed
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During patch recording what is measured?
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The current of a single channel molecule (or more depending upon the density of channels)
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What are the three variations of patch recording?
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1. Whole-cell recording
2. Inside-out patches 3. Outside-out patches |
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Whole cell recording entails what?
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The whole cell is left dangling on the tip of the electrode; there is a large opening at the end of the pipette and if a solution is placed in the pipette it will equilibriate with the cytoplasm of the cell
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What is whole-cell recording useful for?
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Being able to change the internal composition (ions, small molecular weight solutes) by adding a solution to the pipette
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Simply, how do you do "whole-cell recording"?
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Start with a patch clamp, apply suction through pipette until break open the cell so that molecules can flow in and out of the clel
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How can an "inside-out patch" be made?
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Low [Ca2+] makes the membrane break away, leaving the portion of the membrane that is between the microelectrode
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How can an "outside-out patch" be made?
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Pull on cell, it rips and then closes up to protect the hydrophobic lipids
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Why are "inside-out" and "outside-out" patches useful?
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They are small samples of membrane and you can control the environment on each side of the membrane
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What are typical currents through ion channels?
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1-20 picoamps
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When a channel is open, how many ions flow through per second?
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0.6-12 x 10^7 ions /second
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How fast does a typical enzyme handle substrate molecules? What is the fastest enzyme?
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Typical: 10^2 substrate/sec
Fastest: 10^5 substrate/sec |
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Active transport of ions (e.g. by the sodium pump) is how much?
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10^2 - 10^4 / second
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Why must ion channel molecules work by providing a pore through which ions flow through when the channel is open?
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Only way it could be fast enough (on the order of 10^7/sec) vs active transport (on the order of 10^2-10^4/sec)
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What are the four factors that ion channels can be classified based upon?
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1. Conductance
2. Ion Selectivity 3. Gating Properties 4. Pharmacology |
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How can you measure the ion conductance?
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Change the potential across the patch and measure the current that flows when the channel opens. This plot of I vs Vm has a slope that is the conductance
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What is the usual range of conductance, g?
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5-400 pS (picoSiemens)
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Opening an closing of a channel involves a change in what?
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Conformation (3D structure)
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What is the reversal potential?
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Below where the current switches directions; at equilibrium potential
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