Experiment Techniques
Technique used to visualize proteins
Western blot
What kind of molecules can diffuse across the membrane the easiest?
Small and non-polar molecules
What does it mean when a membrane is depolarized?
Membrane potential is less negative
Since electrical signals can't jump the synapse, what is the purpose of neurotransmitters?
Turn an electrical signal into a chemical signal
What kinds of effects do cell signaling pathways lead to?
altered metabolism, altered gene expression, altered cell shape or movement
Technique used to see if proteins are interacting
Co-IP
How many Na+ are let out and how many K+ are brought in?
What allows action potentials to travel in one direction?
Inactivation of voltage gated channels/refractory period
What kind of receptors are primarily found in postsynaptic cells?
Ionotropic receptors
Rank the effects of cell signaling from fastest to slowest
Cell shape/movement, metabolism, gene expression
What 2 techniques are involved in a Co-IP?
SDS PAGE and Western Blot
What is the charge of a resting membrane potential?
Negative
What kinds of ions are excitatory and which are inhibitory?
Na+ and Ca2+ are excitatory and Cl- and K+ are inhibitory
What kinds of receptors do not have their binding site and channel associated with one another?
Metabotropic receptors
List the 3 kinds of receptors
Ion-coupled receptors, GPCR, Enzyme coupled receptors
What is the purpose of the alternative step?
Overexpress a protein of interest so there is more available and is more cost effective
What controls the resting membrane potential?
The sodium/potassium pump and potassium leak channels
What are the steps of an action potential?
1. Resting state
2. Local depolarization
3. Rising phase
4. Falling phase
5. Undershoot
How does cocaine function in the brain?
Inhibits dopamine reuptake transporters so it accumulates in the synapse
The amino acid _____________ acts as a _____________
Aspartic acid, phosphomimetic
List the steps (in order) of a Co-IP
1. Lyse cells.
2. Incubate lysate with antibody to protein
3. Spin down, remove supernatant, rinse breads, spin down again
4. SDS PAGE and western blot for the other protein
What are the steps of the Sodium potassium pump?
1. Pump in E1 conformation, Na+ ions bind to inside
2. Gate closes
3. ATP hydrolysis
4. ADP released, E2 conformation and Na+ released
5. K+ binds inside
6. Pump occluded, Aspartic acid dephosphorylated
7. ATP bound
8. Back to E1 conformation, K+ released
1. VG Ca2+ channel closed on a presynaptic cell
2. Action potential arrival open VG channel, Ca2+ enters cell and helps vessicle w/ neurotransmitters fuse to membrane, neurotransmitters enter synapse
3. Neurotransmitters bind ligand gated channels in postsynaptic cell, channel opens, ions rush and propagate action potential in postsynaptic cell
What are the steps to the propagation of an action potential in the postsynaptic cell?
1. Glutamate binds AMPA and NMDA channels
2. Na+ entry through AMPA depolarizes postsynaptic cell
3. Depolarization ejects Mg2+ from NMDA, channel opens
4. Ca2+ enters through NMDA
5. Ca2+ activates secondary messenger pathway and further depolarizes membrane and action potential is generated
How are G proteins inactivated?
Intrinsic GTP hydrolysis