17. Intracellular Messengers- Part 2: The Role of Calcium in Muscle Contraction

Focus of this lecture?

• Linking muscle cell action potential → actual contraction

What are myofibrils made of, and how is contraction organized in skeletal muscle?

• Myofibrils = bundles of sarcomeres (contractile units).

• Sarcomere structure:

  • Thick filaments: myosin (heavy chains)

  • Thin filaments: actin

• Mechanism of contraction:

  • Myosin heads “walk” along actin → pull Z-lines closer → sarcomere shortens.

  • Multiple sarcomeres in parallel → large-scale muscle contraction.

• Sarcoplasmic reticulum (SR):

  • Specialized ER surrounding sarcomeres.

  • Stores and regulates Ca²⁺ for contraction.

  • Mechanisms of Ca²⁺ release apply to skeletal, cardiac, smooth muscle, and even non-muscle cells with ER.

How does calcium regulate actin-myosin interaction in skeletal muscle contraction?

• Resting state:

  • Myosin heads bound to ADP + Pi; tropomyosin blocks actin binding sites.

• Role of Ca²⁺:

  • Calcium released into cytosol binds troponin (Ca²⁺-binding protein) in actin filament.

  • Troponin undergoes conformational change → moves tropomyosin off actin binding sites.

• Result:

  • Myosin heads bind actin → ATP hydrolysis → power stroke → sarcomere shortens.

• Link: This mechanism connects cytosolic calcium increase to functional muscle contraction.

How do muscle action potentials increase cytoplasmic calcium and why is this important for contraction?

• Depolarization: Muscle action potentials rapidly increase cytosolic Ca²⁺.

• Sarcolemma & organelles:

  • Sarcolemma (cell membrane), mitochondria (ATP supply), and sarcoplasmic reticulum (SR) store Ca²⁺.

  • SR network wraps around sarcomeres to ensure uniform Ca²⁺ release.

• Function:

  • Ca²⁺ binds troponin, moves tropomyosin → exposes actin for myosin binding.

  • ATP from mitochondria needed for myosin power stroke.

  • Without ATP → myosin binds actin but cannot contract → rigor mortis.

• Key point: Efficient Ca²⁺ release and ATP availability are critical for synchronized muscle contraction.

What are the main intracellular calcium release channels and how are they regulated in muscle and non-muscle cells?

• Two main types of Ca²⁺ release channels:

  1. Ryanodine receptors (RyRs) – primarily in sarcoplasmic reticulum (muscle cells).

  2. IP3 receptors (IP3Rs) – found in SR (muscle) and ER (non-muscle cells).

• Activation mechanisms:

  • RyRs: triggered by voltage changes or Ca²⁺-induced Ca²⁺ release (CICR).

  • IP3Rs: activated by IP3, produced via Gq protein → PLC → PIP2 → DAG + IP3.

• Calcium flow: IP3 can diffuse through cytosol to bind IP3Rs; DAG remains membrane-bound.

• Outcome: Opening of these channels rapidly increases cytosolic Ca²⁺ for contraction or signaling.

How do IP3 receptors mediate calcium release from the sarcoplasmic reticulum, and what role does calcium itself play in this process?

• IP3 receptor (IP3R): a ligand-gated calcium channel (ionotropic) in the sarcoplasmic reticulum.

• Activation: IP3 binds to the receptor → opens channel → releases Ca²⁺ into cytosol.

  • Positive feedback: low cytosolic Ca²⁺ enhances IP3R opening when IP3 binds → amplifies calcium release.

  • Negative feedback: high cytosolic Ca²⁺ inhibits IP3R to prevent excessive Ca²⁺ release.

• Calcium binding sites: 4 sites on IP3R; calcium modulates receptor sensitivity.

• Functional effect: presence of Ca²⁺ increases receptor sensitivity to IP3 (Ca²⁺ binds to receptor but doesn’t open it) → less IP3 needed for channel opening → allows rapid, regulated Ca²⁺ signaling.

What are the hierarchical levels of calcium release from internal stores, and how do they depend on IP3 concentration?

• Internal calcium stores: sarcoplasmic reticulum (muscle) or endoplasmic reticulum (non-muscle).

• Blips:

  • Calcium release from single IP3 receptor channels.

  • Triggered by low IP3 levels (~10–30 nM).

  • Represents minimal, localized Ca²⁺ signaling.

• Puffs:

  • Calcium release from clusters of neighboring IP3 receptors.

  • Triggered by higher or more sustained IP3 levels (~50–600 nM); duration of ~ 1s; size of 6μm

  • Produces larger, localized Ca²⁺ signals across the ER/SR.

• Functional significance: hierarchical release allows graded, spatially controlled calcium signaling.

How do calcium waves form from IP3 receptor activation, and what is the role of positive and negative feedback?

• Calcium-induced calcium release (CICR):

  • Low cytosolic Ca²⁺ → potentiates IP3 receptors (or ryanodine receptors), increasing calcium release.

  • High cytosolic Ca²⁺ → inhibits further release to prevent overactivation.

• Mechanism of a calcium wave:

  • Local calcium release (puff) raises cytosolic Ca²⁺.

  • High Ca²⁺ at the release site negatively feeds back to inactivate those receptors.

  • Low Ca²⁺ at the periphery positively potentiates neighboring receptors, making them more sensitive to IP3.

  • Sequential activation propagates a wave of calcium release across the SR/ER.

• Functional significance: ensures synchronized, directional contraction rather than random, patchy activation.

How is intracellular Ca²⁺ signaling observed?

• Visualization:

  • Use fluorescent Ca²⁺ probes that fluoresce when bound to free cytosolic calcium.

  • Blips: tiny, local calcium releases (initial “lightning”).

  • Puffs: larger, clustered releases from multiple IP3 receptors.

  • Waves: coordinated propagation of calcium across the cell.

• Functional significance:

  • In muscle cells, waves allow sequential, coordinated contraction along the length of the fiber.

  • Ensures efficient and organized contraction rather than random or patchy activation of sarcomeres.

How does the structure of the sarcoplasmic reticulum and T-tubules facilitate calcium release in skeletal muscle?

• Sarcoplasmic reticulum (SR): stores very high concentrations of Ca²⁺, positioned near sarcomeres.

• T-tubules: deep invaginations of the sarcolemma that transmit surface action potentials deep into the muscle fiber.

• Triad: each T-tubule flanked by two terminal cisternae of SR, forming a structural unit for efficient Ca²⁺ signaling.

• Electrical isolation: SR membrane is not directly depolarized by the T-tubule AP.

• Solution – indirect coupling: physically interacting channels between T-tubules (DHPR) and SR (RyR) translate membrane depolarization into Ca²⁺ release from SR.

What are DHPR channels, and how do they interact with RyR in skeletal muscle excitation-contraction coupling?

• DHPR (dihydropyridine receptors): L-type voltage-gated calcium channels located in T-tubules; act as voltage sensors.

• RyR (ryanodine receptors): calcium-release channels in the sarcoplasmic reticulum (SR).

• Indirect coupling: At rest, DHPR physically blocks RyR; T-tubule depolarization triggers conformational change in DHPR, opening RyR → Ca²⁺ release from SR.

• Isoforms and evolution: Different RyR isoforms exist in skeletal, cardiac, and smooth muscle; some species (e.g., billfish) evolve RyR to create specialized functions (e.g., heater muscles) instead of contraction.

• Key concept: This system links action potentials in the membrane to SR calcium release without direct electrical propagation.

What are the key structural and functional features of DHPR (L-type) calcium channels in skeletal muscle?

• Type: L-type (long-lasting) voltage-gated calcium channels in T-tubules of skeletal muscle.

• Function: Serve as voltage sensors for excitation-contraction coupling; detect action potential depolarization.

• Voltage sensor: Segment S4 responds to membrane potential changes.

• Selectivity pore: Segments S5–S6 form the Ca²⁺-selective channel.

• Role in muscle: Rapid opening upon depolarization → triggers RyR opening in SR → Ca²⁺ release → contraction.

• Significance: Enables high-frequency excitation-contraction-relaxation cycles in skeletal muscle.

How do DHPR and ryanodine receptors work together to mediate calcium release during skeletal muscle depolarization?

• Resting state: DHPR (L-type Ca²⁺ channel) has a long cytoplasmic extension that physically blocks RyR, preventing Ca²⁺ leak from SR.

• Depolarization: AP travels down T-tubules → activates DHPR voltage sensor (S4 segment).

• DHPR response:

  • Opens its own small Ca²⁺ channel → minor Ca²⁺ influx from extracellular space.

  • Mechanically unblocks RyR → Ca²⁺ released from SR.

• Calcium-induced calcium release (CICR): Incoming Ca²⁺ further activates RyR → amplifies Ca²⁺ release.

• Result: Rapid, large increase in cytosolic Ca²⁺ (mostly from SR) → triggers muscle contraction.

Why is sarcoplasmic reticulum (SR) calcium more critical than extracellular calcium for skeletal muscle contraction?

• Calcium sources comparison:

  • SR Ca²⁺ release: Major contributor to cytosolic Ca²⁺, drives muscle contraction efficiently.

  • Extracellular Ca²⁺ via DHPR: Small contribution, insufficient alone to trigger full contraction.

• Experimental insight: Blocking extracellular Ca²⁺ (DHPR) still allows full contraction via SR; blocking SR Ca²⁺ prevents effective contraction even with extracellular Ca²⁺.

How does an action potential in a motor neuron trigger skeletal muscle contraction?

1. Neuron depolarization: AP reaches presynaptic terminal → opens voltage-gated Ca²⁺ channels → Ca²⁺ influx.

2. Neurotransmitter release: Calcium signals vesicles to release acetylcholine (ACh) into the synaptic cleft.

3. Muscle endplate activation: ACh binds nicotinic ionotropic receptors → Na⁺ influx → endplate potential → triggers voltage-gated Na⁺ channels at periphery of end plate.

4. Intracellular Ca²⁺ signaling:

   • Small Ca²⁺ release via IP3 receptors from sarcoplasmic reticulum (SR) potentiates neighboring IP3 receptors and opens RyR.

   • AP propagates down T-tubules → DHPR (voltage-gated L-type Ca²⁺ channel) opens → mechanically unblocks RyR → large Ca²⁺ release from SR.

5. Muscle contraction machinery:

   • Cytosolic Ca²⁺ binds troponin → shifts tropomyosin → exposes myosin-binding sites on actin.

   • Myosin heads hydrolyze ATP → perform power stroke → sarcomere shortens → muscle contraction.

How does muscle relaxation occur after contraction, and why is rapid calcium reuptake important?

1. Muscle relaxation requirement: After contraction, cytosolic Ca²⁺ must be removed so tropomyosin can block myosin-binding sites → sarcomeres relax.

2. SERCA pumps:

   • Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA) actively transport Ca²⁺ back into the sarcoplasmic reticulum.

   • This process consumes significant ATP, making relaxation energy-intensive.

3. Rhythmic contractions: Rapid Ca²⁺ clearance enables quick sequential contraction-relaxation cycles, crucial for high-performance muscles and rhythmic activities.

4. Evolutionary/functional examples:

   • Billfish “heater” muscle: Leaky ryanodine receptors cause futile Ca²⁺ cycling → SERCA works harder → ATP consumption generates heat.

   • Athletic skeletal muscle: Efficient SERCA function allows fast, repeated contractions → supports endurance and high-speed movements.

What are the main mechanisms cells use to maintain calcium homeostasis and promote muscle relaxation?

1. SR / ER mechanisms:

   • SERCA pumps: Actively transport Ca²⁺ from cytosol back into SR/ER using ATP.

   • IP3 receptors & ryanodine receptors: Release Ca²⁺ from SR/ER in response to signaling; regulated by positive/negative feedback.

2. Plasma membrane mechanisms:

   • PMCA (Plasma Membrane Ca²⁺ ATPase): Pumps Ca²⁺ out of the cell using ATP.

   • Na⁺/Ca²⁺ exchanger (NCX): Secondary active transport; moves 1 Ca²⁺ out for 3 Na⁺ in, using Na⁺ gradient.

   • Ca²⁺-activated K⁺ and Cl⁻ channels: Indirectly influence Ca²⁺ homeostasis by restoring membrane potential after action potentials (not on the midterm!)

3. Integration with signaling pathways:

   • G protein-coupled receptors (GPCRs) can activate voltage-gated Ca²⁺ channels and prime IP3/ryanodine receptors for coordinated Ca²⁺ release.