Vinberg

Question 1

Second messenger:

Answer 1

A diffusable signaling molecule that is rapidly produced/secreted, which can then go on to activate effector proteins within the cell to exert a cellular response.

Question 2

Signal Cacade

Answer 2

Signal –> Receptor –> 2nd messenger –> effector molecule –> biological response

Question 3

Why Ca2+ and why it is a great messenger? ( 5 reasons)

Answer 3

1. Abundance and availability. The 5th most abundant element on Earth
2. Does not have to be made, cannot be destroyed
3. Can be exchanged quickly - microseconds (large transmembrane gradient)!
4. Biophysical properties: Ca charge & size regulate protein charge and size
5. Range: hundreds of proteins adapted to bind Ca from nM to mM

Question 4

Why is Ca2+ an ideal carrier of information?

Answer 4

very high signal to noise (SNR) ratio

The resting values are between 30-100 nM,
which means that RMS noise is in the range
of 5-10 nM. Local Ca2+ can easily go to ~1
μM:

[Ca2+]i changes from ~20 nM to 150 - 300 nM, and locally can increase to 1 –
100 μM

Question 5

Calcium is taken out of the cytosol by three types
of clearance mechanisms

Answer 5

1. Pumps (use ATP)
2. Exchangers (use Na gradient)
3. Mitochondrial Uniporters (passive flux)

Question 6

Where does the energy come from to maintain
low cytosolic [Ca2+]?

Answer 6

ATP, Exchangers (ionic gradient) as well as electron train

Question 7

GCaMP

Answer 7

combination of calmodulin (Ca-binding) and GFP.

unbound calmodulin quenches fluorescence of GFP

Question 8

evolutionary constraint of Ca2+ as a signaling molecule

Answer 8

Ca2+ precipitates phosphates

Question 9

Why did evolution favor cells that
could maintain low cytosolic
Ca2+?

Answer 9

Cells w/ low [Ca2+] didn’t create phosphate precipitates that would have otherwise been lethal to cell

Question 10

Why low cytosolic Ca2+ may be critical in making Ca2+
a good intracellular signaling
molecule?

Answer 10

creates a large gradient across cell membrane

Question 11

Answer 11

Question 12

Answer 12

Question 13

Why Ca2+ diffuses passively into mitochondria
even with much higher Ca2+ in the mitochondria
than in cytosol?

Answer 13

this is due to the proton gradient. inner-mitochondria is much much more negatively charged. electrical force that is driving this

Question 14

Answer 14

Question 15

who do some Ca2+ channels have different electric behvaior?

Answer 15

alternative splicing in different tissues. Some exons are not included in mRNA

Question 16

Specificity and versatility by local Ca2+ regulation:
How do you make a Ca2+ compartment?

Answer 16

1. Anatomy
2. Diffusion
   3.Spatially regulate influx, clearance, sequestration
   4.Spatially distribute the targets – hundreds of Ca2+ sensors

Question 17

Why should KD of the
buffer roughly match free
Ca2+ in the compartment?

Answer 17

This calculation shows that when [Ca2+]free is equal to KD, half of the Ca2+ buffer has Ca2+ bound to it.
If KD»[Ca2+]free (low affinity buffer), none of the buffer is bound to Ca2+
If KD«[Ca2+]free (high affinity buffer), every buffer molecule occupies Ca2+ (saturation)
This is why it is important to match the free Ca2+ concentration with KD of Ca2+ fluorescence dye (e.g. gcamp)

Question 18

Rule of Thumb II:

Answer 18

a mobile buffer will tend to disperse domains of elevated
Ca whereas a fixed buffer will tend to prolong them

Question 19

Why is rule of thumb II true?

Answer 19

if buffer is mobile. it binds calcium, then diffuses away to another part of the cell. In this new area of the cell, the concentration of free calcium is less than the old area, which causes the calcium to unbind from the buffer. This mobile buffer moved calcium from an area of high calcium to low calcium

with fixed buffer near influx of Ca. buffers uptake a lot of Ca, this enables more Ca to flow into cell (flux is dependent on electrochemical gradient). once channel is closed, more Ca has entered the cell, thus prolonging the period of high Calcium

Question 20

Neuronal ER + 2 important functions

Answer 20

• Ca2+ homeostasis, synaptic transmission & plasticity
• A single and continuous organelle: important for protein
  transport and propagation of Ca signals

Question 21

Why spines with ER have larger Ca2+ signals?

Answer 21

ER stores a large amount of Ca

• Generation of Ca2+ signals and control of Ca2+ homeostasis
• Calcium-Induced Calcium Release
• Store-Operated Calcium Entry
• Ca2+ waves/oscillation
• Communication with e.g. mitochondria

Question 22

Ryanodine receptor

Answer 22

RyRs act as intracellular amplifiers(Calcium-
calcium induced calcium release

Question 23

IP3 receptors are activated by what?

Answer 23

Activated by metabotropic signaling via Gq/11 proteins

Question 24

Answer 24

Question 25

Answer 25

Question 26

IP3 receptors are _____

Answer 26

coincidence detectors

Question 27

unfolded protein response

Answer 27

Maintaining high ER Ca2+ levels is essential for cellular survival.

Question 28

current SOCE (store operated calcium entry) model

Answer 28

Question 29

Summary of lecture 2

Answer 29

• The ER is dynamic and active.
• Fills the entire neuron
• Communicates with the plasma membrane and mitochondria
• Buffering & Tunneling
• Different types of Ca release channels; coincidence detectors & amplifiers
• STIM1 as sensors of store depletion/ Orai as store-operated channel

Question 30

LTP model w/ Ca

Answer 30

synaptic activity
↑submembrane Ca2+/CaM
↑Ca2+/CaM kinase activity
↑pCREB, ↑pAMPAR
long-term plasticity

Question 31

If EGTA blocks CREB activation,

Answer 31

global Ca2+ change.

Question 32

If BAPTA blocks CREB activation,

Answer 32

local Ca2+ change.