Metabotropic Receptors- Biochemical Cascades

Question 1

Calcium and Calmodulin:

Answer 1

• At rest, concentration of free cytoplasmic calcium is maintained by Ca2+-Mg2+ATPases (calcium pumps) that transport ions out of the cell or inter the ER for storage
• Cytoplasmic calcium levels can be rapidly elevated through the activation of voltage gated calcium channels or ionotropic receptors like the NMDA receptor
○ Voltage gated calcium channels also found on the soma and dendrites, not just the synaptic terminal
• Ca2+ (second messenger) interacts with an intracellular Ca2+ binding protein, calmodulin to regulate a number of proteins
○ On its on, calcium isn’t a second messenger- needs to be bound to calmodulin
Calcium bound calmodulin is the second messenger (CaM)
Activates CaM-K II- Calcium-Calmodulin protein Kinase II

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Question 2

PDE inhibitor

Answer 2

• PDE (phosphodiesterase) inhibitors- enhance effects of cAMP or cGMP by inhibiting their degradation
i.e. viagra is a PDE inhibitor

Question 3

Mechanism of cAMP & cGMP:

Answer 3

• Have similar mechanisms of action, but cGMP can be activated in more than one way
• Ligand binds, activates cAMP/cGMP, cAMP/cGMP activates ion channels, PKAs…etc
• cGMP is not always membrane bound (some free floating), can be stimulated with NO pathway as well
○ Ligand binds to calcium receptor, calcium levels increase
○ Increased calcium levels causes nitric oxide synthase (NOS) to synthesize nitric oxide (NO)
NO activates guanylyl cyclase, creates cGMP

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Question 4

Immediate- Early Genes (IEG)

Answer 4

• Second messengers can impact transcription factors
• cAMP activated PKA phosphorylates transcription factors (cAMP response element binding proteins- CREB), which binds to promoter regions (cAMP response element- CRE) on DNA to promote transcription of IEGs
• IEGS are the first phase of gene activation, doesn’t result in lasting structural change, but acts as the next step in a process that may result in lasting change
• Inactivation of CREB impairs long term memory
Impairs synaptic plasticity, impairs learning

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Question 5

Late Response Genes:

Answer 5

• CREB (and other transcription factors) induce a group of IEGS that include
○ C-fos, c-jun, fos B, jun B, zif-268
• Protein products of these IEGs are transcription factors (Fos, Jun) which regulate the expression of other genes (late response genes)
• Late response genes result in lasting changes
mRNA is induced rapidly (within 15 minutes) but only transiently (only remains elevated for 30-60 minutes, doesn’t last long)

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Question 6

Fos Protein Imaging:

Answer 6

• Interested in measuring levels of c-fos gene product in brain regions important for object recognition (perirhinal cortex)
• Rats are shown a novel object, over time, the novel object becomes familiar
• On test day, one eye is exposed to the familiar object, the other eye is exposed to the novel object
○ Demonstrates within subject design
• Rats sacrificed one hour after exposure to object, brain tissue was stained to look for an antibody that binds to the cfos protein
• Found that there were higher levels of fos in the hemisphere exposed to unfamiliar stimuli than hemisphere exposed to familiar object
More cells fire, and fire more often with a novel object than a familiar one

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Question 7

Zif268 Study:

Answer 7

• Studied tissues in a petri dish, looking at zif268 protein (more specific than c-fos, zif268 is upregulated in synapses that are recently activated in high frequencies, important for forming LTP)
• If zif 268 is knocked out, LTP is not formed
• Hippocampus undergoes high frequency stimulation (induces LTP)
Found that both mRNA and protein product of zif268 levels increased

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