Calcium Signaling

Question 1

1. Describe the functions of cytoplasmic Ca2+ ion buffers and how these buffers affect cytoplasmic Ca2+ signals.

Answer 1

• Ca doesn’t need enzymes but rather just enters Ca from sources (ER/SR, nuclear envelope, mt, and extracellular Ca) and leaves into sinks (same things).
• Cellular Ca buffers are proteins that bind Ca and help with Ca signaling.
• Buffers in cytoplasm (like parvalbumin) restrict the spatial and temporal spread of Ca and serve as temporary storage site for Ca while the slow transport processes are operating –> create distinct signaling domains.
• In ER/SR lumen, high-capacity, low affinity buffers (like calsequestrin) allow large quantities of Ca to be stored without generation of a large gradient in the concentration of free Ca.

Question 2

2. Describe the routes by which extracellular Ca2+ enters the cytoplasm, the routes by which Ca2+ moves out of the ER/SR into the cytoplasm, and the routes by which Ca2+ is extruded from the cytoplasm (a) into the extracellular space and (b) into the lumen of the ER/SR.

Answer 2

-From sources –> Ca Ion channels (passive, electrochemically downhill)
-plasma membrane has VG (when depolarized) and LG (nACh or glutamate)
-Ca channels, as well as store-operated Ca channels (like Orai1; depends on depletion of Ca from ER/SR to work)–> from outside cell into cytoplasm
-ER/SR (and nuclear envelope) –> IP3 receptors (as cleaved from PLC?),
RyR’s –> Ca moves from lumen into cytoplasm.
-mt has a uniporter and a permeability transition pore (but direction depends on Ca gradient)

Into sinks –> uses transporters (active, against electrochemical gradient, slower than ion channels)

• Ca pumps use ATP to move Ca into ECF or into lumen of ER/SR (like SERCA pumps).
• SERCa ATPase at ER/SR and PMCa ATPase at plasma membrane (gets Ca into ECF).
• Na/Ca exchangers move Ca to ECF or into mt (3Na for 1Ca) –> derives energy from Na.
• mt uniporter can move Ca into mt if gradient favors this

Question 3

3. Describe EF hands and C2 domains, identify the archetypical protein that contains EF hands and the archetypical protein that contains a C2 domain, and determine whether these domains are present in other proteins.

Answer 3

Effectors can impact:

• surface membrane potential (Ca channels and Ca-activated channels –>neuronal bursting and cardiac pacemaking)
• PKC (translocation to membrane)
• synaptotagmin (Ca-dependent fusion of synaptic vesicles)
• calmodulin (multiple downstream targets)
• bind of Ca causes C2 domains to associate with plasma membrane:
• in PKC, there are C2 domains that influence phosphatidylserine (?) and cause translocation to membrane
• in synaptotagmin, there are also C2 domains that Ca binds to activate the protein (can bind 3 Ca ions)
• **so these C2 domains are heavily conserved
• Calmodulin is also strongly conserved–> contains 4 “EF-hands” that are Ca binding sites, and each Ca molecule coordinated with 5 oxygens.
• Calmodulin binds to and confers Ca regulation to many other proteins (like ion channels, kinases, phosphatases, PDEs) –> EF hand in calmodulin found in lots of other Ca effectors (like parvalbumin, calpain, and troponin).
• can have Ca-binding motifs that are not like C2 or EF-hands.

How does Ca trigger both contraction and relaxation?

• maintained depol. activates VG Ca channels –> triggers release of Ca from nearby RyR’s –> causes global Ca increase and smooth muscle contraction
• RyR causes large localized Ca increase –> causes hyperpolarization via activation of Ca-activated K+ channel –> closes VG Ca channels and causes smooth muscle relaxation

Ca and helper T-lymphocytes:

• peptide/MHC complex binds T-cell receptor’s membrane
  
  • TCR’s aggregate –> activation of Tyr kinase
  • this activates PLC –> cleaves PIP2 into IP3 (which activates IP3R’s in ER and depletion of ER Ca store) and DAG.
  • a store-operated Ca channel in the plasma membrane gets activated (SOC), causes Ca influx into cell. Ca-calmodulin binds calcineurin, which can dephosphorylate NFAT –>: goes to nucleus and increases transcription of IL-2
• Mutations in RyR2 (cardiac) causes familial polymorphic ventricular tachycardia.
• Ca release via RyR2 generally synchronized with Ca entering during cardiac AP.
• Mutations –> delayed release –>delayed depolarization via Na/Ca exchanger –> arrhythmia.