20. Homeostasis: Regulation of Intracellular Calcium

How does calcium function as an ON/OFF switch in muscle and other cells?

• ON reaction: Ca²⁺ floods into cytoplasm → binds effector proteins (e.g., troponin) → exposes myosin binding sites → triggers contraction or other Ca²⁺-activated pathways.

• OFF reaction: Ca²⁺ must be rapidly removed so it dissociates from effectors:

  • PMCA pumps Ca²⁺ out of the cell (ATP-dependent).

  • Na⁺/Ca²⁺ exchanger exports Ca²⁺ via secondary active transport.

  • SERCA returns Ca²⁺ to the SR.

• Ca²⁺ is not degraded, only sequestered into compartments where it can’t activate proteins.

• Muscle function depends on Ca²⁺ cycling: brief cytoplasmic spikes (ON) followed by rapid clearance (OFF).

• This fast ON/OFF cycling enables repeated, coordinated contractions across whole muscles.

How does the mitochondrial calcium uniporter (MCU) complex regulate Ca²⁺ entry into the matrix?

• Resting cytoplasmic Ca²⁺ ~100 nM (very low).

• High Ca²⁺ stores:

  • Extracellular fluid: 1–2 mM

  • SR/ER: very high

  • Mitochondria: high (similar to SR)

• Low Ca²⁺ areas: cytoplasm, nucleus.

• Key Ca²⁺ regulators:

  • VG Ca²⁺ channels → Ca²⁺ influx.

  • PMCA → pumps Ca²⁺ out (ATP-dependent).

  • Na⁺/Ca²⁺ exchanger → secondary active export.

  • IP₃R & RyR → Ca²⁺ release from SR/ER.

  • SERCA → returns Ca²⁺ to SR/ER.

• Homeostasis must support rapid spikes (ON) and rapid clearance (OFF) while maintaining stores for future signaling.

What roles do calmodulin and Ca²⁺-buffering proteins play in calcium signaling?

• Calmodulin:

  • Monomer with two globular domains + hinge.

  • Ca²⁺ binding → conformational change → wraps around target proteins → alters the protein activity.

• Ca²⁺ buffer proteins (e.g., calbindin, parvalbumin):

  • Bind Ca²⁺ quickly to reduce free Ca²⁺.

  • Temporarily block Ca²⁺ from activating effectors.

  • Speed the OFF reaction by limiting Ca²⁺ availability before pumps fully clear it.

How does calmodulin regulate PMCA to control Ca²⁺ clearance?

• PMCA pumps Ca²⁺ out of the cell but is autoinhibited at rest to avoid wasting ATP.

• High cytosolic Ca²⁺ → Ca²⁺ binds calmodulin → Ca²⁺/CaM complex binds PMCA’s inhibitory domain.

• This removes autoinhibition → activates PMCA → rapid Ca²⁺ export.

• As Ca²⁺ falls, Ca²⁺ unbinds calmodulin → PMCA becomes autoinhibited again.

• Creates a feedback system: PMCA turns on only when Ca²⁺ is high.

How does the Na⁺/Ca²⁺ exchanger contribute to Ca²⁺ clearance?

• Secondary active transport: uses Na⁺ gradient (maintained by Na⁺/K⁺-ATPase) to export Ca²⁺.

• Fast, always running in the background—no calmodulin required.

• Critical in presynaptic terminals, where Ca²⁺ must be cleared quickly to stop continuous vesicle fusion.

• Prevents excess transmitter release by removing Ca²⁺ after each action potential.

Why does Ca²⁺ flow into mitochondria, and what drives it?

• Mitochondria have two membranes; Ca²⁺ passes outer membrane via VDAC channels.

• Inner membrane Ca²⁺ entry is driven by the strongly negative mitochondrial matrix (~–200 mV).

What makes the inner mitochondrial matrix so negative?

• This negativity results from the electron transport chain pumping protons into the intermembrane space.

• No pumps required: Ca²⁺ moves via channels because of the large electrical gradient.

• Mitochondria act as a secondary Ca²⁺ reservoir and use Ca²⁺ to stimulate metabolic enzymes.

How does the mitochondrial calcium uniporter (MCU) complex regulate Ca²⁺ entry into the matrix?

• MCU (inner membrane channel) allows Ca²⁺ entry when open.

• EMRE (MCU regulator) organizes the complex.

• MICU1/2 are Ca²⁺-binding gatekeepers:

  • Low Ca²⁺ → MCU closed.

  • High Ca²⁺ → MICU1/2 bind Ca²⁺ → conformational change → MCU opens.

• Allows controlled Ca²⁺ entry despite huge electrical driving force.

• Mitochondria store Ca²⁺ and also use it to stimulate the Krebs cycle & ETC.

What happens when mitochondria take up too much Ca²⁺?

• Excess matrix Ca²⁺ → overactivation of Krebs cycle + ETC → excessive proton pumping.

• Leads to ROS (reactive oxygen species) production at the electron transport system.

• ROS damage:

  • DNA, proteins, lipids (especially phospholipid inner membrane).

  • Causes cytochrome c leakage, triggering apoptosis.

• Mitochondrial Ca²⁺ overload = major trigger for cell death.

How do mitochondria and ER/SR share Ca²⁺, and why does it matter?

• Linked via MAMs (mitochondria-associated membranes) → direct Ca²⁺ and stress signal transfer.

• Mitochondria act as a reserve reservoir:

  • Can return Ca²⁺ to ER/SR when stores run low.

• However, ROS or Ca²⁺ overload in mitochondria can spread stress to ER/SR → ER stress → apoptosis.

• MAMs allow both beneficial Ca²⁺ sharing and propagation of damage.

Why does reperfusion after ischemia cause dangerous mitochondrial Ca²⁺ overload?

• During ischemia/hypoxia (low O₂):

  • ETC stops → no proton gradient.

  • Ca²⁺ pumps (PMCA, SERCA) fail due to low ATP.

  • Na⁺/Ca²⁺ exchanger fails because it relies on the gradients set up by Na⁺/K⁺ pump, which uses ATP.

  • Ca²⁺ builds up in cytoplasm but does not enter mitochondria (no negative matrix).

• During reperfusion (O₂ returns):

  • ETC restarts instantly → matrix becomes highly negative before Ca²⁺ pumps recover.

  • Massive Ca²⁺ rushes into mitochondria → ROS burst → membrane damage → cytochrome c release → apoptosis.

• This is why reperfusion injury is worse than the ischemia itself.

How do mitochondria safely store Ca²⁺ without triggering damage?

• Mitochondria import inorganic phosphate (Pi).

• High Ca²⁺ + Pi → form calcium phosphate precipitates.

• Precipitated Ca²⁺ = inactive, preventing:

  • Excess Krebs cycle stimulation

  • Excess ETC flux

  • ROS generation

• When ER/SR needs Ca²⁺, free Ca²⁺ diffuses out → due to low Ca²⁺ in mitochondria, precipitates dissolve → replenish Ca²⁺ stores.

• Buffering prevents mitochondrial Ca²⁺ overload except under extreme stress (e.g., ischemia/reperfusion).