Excitation–Contraction Coupling & Calcium Regulation in Skeletal Muscle

Overview & Scope

• Focus: Week 5 – Excitation-Contraction Coupling (ECC) in skeletal muscle
• Builds on prior weeks
  • Week 3 = Cross-bridge mechanics
  • Week 4 = Action potential (AP) propagation + Neuromuscular junction (NMJ)
• Central theme: “How does an electrical event at an α-motor neuron become mechanical force, and how is it stopped just as rapidly?”
• Key numerical references
  • Resting membrane potential (RMP): $V_m \approx -90\ \text{mV}$
  • Peak AP in muscle/T-tubule: $\approx +30\ \text{mV}$

Learning Objectives

• Describe the architecture of the Transverse-tubule (T-tubule) system and the Sarcoplasmic Reticulum (SR).
• Locate & explain operation of voltage-gated Ca²⁺ channels (DHP in T-tubule membrane, mechanically linked to RyR in SR).
• Locate & explain function of Ca²⁺ ATPase pumps (SERCA) in SR membrane.
• Integrate channel & pump activity to show how cytosolic $[Ca^{2+}]$ is raised to start, and lowered to stop, force.
• Sequence the events that link (a) muscle-fiber excitation → force and (b) fiber repolarization → relaxation.
• Trace events from α-motor-neuron AP through muscle relaxation.
• Detail tropomyosin movement and its effect on cross-bridge site exposure.
• Explain how Ca²⁺ binding/unbinding to troponin C (TnC) changes troponin shape and tropomyosin position.

Review: The Cross-Bridge Cycle (Week 3)

1. Attachment – Energized myosin head (ADP·Pi bound) weakly docks to actin.
2. Power stroke initiation – Pi release → head bends ≈ pivot; affinity ↑.
3. Power stroke – Actin filament slides; ADP leaves.
4. Detachment – New ATP binds myosin → actin affinity ↓ → cross-bridge breaks.
5. Return (Cocking) – ATP hydrolysis (ATP → ADP + Pi) re-energizes head, restoring pre-stroke angle.

• Cycle repeats while
  • ATP is available, and
  • Actin sites are exposed (high $[Ca^{2+}]_i$).

How Do We Turn the Cycle OFF? – Metaphors Explored

• Waiting for ATP to deplete? ( leads to rigor mortis; not physiological)
• Prevent filament overlap? ( impractical; sarcomere geometry is fixed)
• Real answer: “Hide-and-seek” – hide actin’s myosin-binding sites by shifting regulatory proteins.

Thin-Filament Regulation

Structural Players

• Actin
  • Monomer = G-actin; polymer = F-actin helix
• Tropomyosin (Tm)
  • Two-stranded α-helical rod; spans 7 successive actins
  • Runs in the groove of F-actin
• Troponin complex (Tn) – globular, 3 subunits
  • TnT – tethers complex to tropomyosin (“T for Tm”)
  • TnI – binds actin & inhibits myosin ATPase (“I for inhibitory”)
  • TnC – Ca²⁺-binding subunit; the molecular switch

Ca²⁺-Dependent Conformations

• Low $[Ca^{2+}]_i$ (~10⁻⁷ M)
  • Ca²⁺ absent from TnC
  • TnI holds Tm over myosin-binding sites ⇒ sites covered, no force
• High $[Ca^{2+}]_i$ (~10⁻⁵ M)
  • Ca²⁺ binds TnC (4 Ca²⁺/TnC)
  • Troponin changes shape → pulls tropomyosin deeper into actin groove
  • Binding sites exposed → cross-bridge cycling & force

Myocyte Ultrastructure: T-Tubules & SR

T-Tubules (Transverse Tubules)

• Invaginations of sarcolemma that penetrate at each A-I junction.
• Wrap 360° around every myofibril; lumen is extracellular space.
• Function: Conduct the surface AP deep so all sarcomeres are synchronously activated (rapid <1 ms).

Sarcoplasmic Reticulum (SR)

• Specialized smooth ER; major Ca²⁺ store.
• “Lacy sleeves” around each myofibril; bulges called Terminal Cisternae abut each T-tubule.
• T-tubule + 2 terminal cisternae = Triad.

Molecular Junction in the Triad

• DHP receptor (DHPR)
  • A L-type voltage-sensitive Ca²⁺ channel embedded in T-tubule membrane.
  • In skeletal muscle functions mainly as voltage sensor (not as Ca²⁺ conduit).
• Ryanodine Receptor (RyR1)
  • Large Ca²⁺-release channel in SR membrane, directly apposed to DHPR.
  • Mechanical coupling: depolarization-induced conformational change in DHPR pulls open RyR.

Ca²⁺ Pumps – SERCA

• Sarco/endoplasmic reticulum Ca²⁺-ATPase located throughout SR membrane.
• Uses 1 ATP to transport 2 Ca²⁺ from cytosol → SR lumen.
• High turnover; lowers $[Ca^{2+}]_i$ back toward resting $\approx 10^{-7}\,\text{M}$ within $\sim 30\,\text{ms}$ after AP.
• Pump rate proportional to $[Ca^{2+}]_i$ (self-limiting: faster when Ca high).

Excitation–Contraction Coupling: Event‐by‐Event

(i) From α-Motor Neuron AP → Force

1. AP travels along α-motor neuron axon (saltatory, myelinated).
2. Reaches terminal bouton; triggers Ca²⁺ influx → ACh exocytosis.
3. ACh crosses NMJ cleft ((~50 nm)) → binds nicotinic AChRs on motor-end-plate.
4. End-plate potential (EPP); if threshold met, muscle sarcolemma fires an AP.
5. AP propagates along surface & dives into T-tubules.
6. Depolarization (+30 mV) alters DHPR conformation → opens RyR in SR.
7. Ca²⁺ floods out of SR (down huge gradient: lumen ≈ $1\,\text{mM}$ vs cytosol ≈ $0.1\,\mu\text{M}$).
8. $[Ca^{2+}]_i$ rises to $\sim 10^{-5}\,\text{M}$ within <2\,\text{ms}.
9. Ca²⁺ binds TnC → tropomyosin shifts → actin sites revealed.
10. Cross-bridge cycling proceeds, generating tension/shortening.

(ii) Repolarization → Relaxation

1. Sarcolemma & T-tubules repolarize (Na⁺ channels inactivate, K⁺ outflow).
2. DHPR returns to resting state → RyR closes.
3. SERCA pumps dominate → Ca²⁺ resequestered into SR.
4. $[Ca^{2+}]_i$ falls below threshold; Ca²⁺ dissociates from TnC.
5. Troponin/Tropomyosin complex returns to blocking position.
6. Myosin can no longer bind actin → cross-bridge cycling ceases.
7. Elastic elements & antagonist muscles restore length → relaxation.

Integrated Roles of Channels & Pumps in $[Ca^{2+}]$ Regulation

• Voltage-gated release (RyR) causes rapid, massive Ca²⁺ spike → force.
• Ca²⁺ ATPase (SERCA) ensures equally rapid decline once release stops.
• The time course of $[Ca^{2+}]_i$ therefore mirrors the twitch:
  • Rise phase $\approx 2\,\text{ms}$ (release > uptake)
  • Peak $\approx 5\,\text{ms}$
  • Decline $\approx 30\,\text{ms}$ (uptake > release)
• Force production is proportional to the fraction of troponin molecules with bound Ca²⁺.

Visual/Conceptual Aids Recalled in Lecture

• Animations of T-tubule depolarization waves and Ca²⁺ flashes – illustrate why internal sarcomeres need T-tubules for simultaneity.
• “An exciting couple of contractions” slides: show successive frames—RMP, depolarized membrane, RyR open, Ca²⁺ binding, pump futile while channel open, repolarization, pump dominance.
• Infographic: “A User’s Guide to Muscles” by Eleanor Lutz – reinforces terminology (actin, myosin, ACh, ATP, mitochondria).
• Hide-and-seek metaphor for thin-filament regulation.

Practical, Clinical, Ethical Notes

• Botulism toxin blocks ACh release → flaccid paralysis (failure of step 2 above).
• Malignant hyperthermia – RyR mutation; excessive Ca²⁺ release, sustained contraction, heat.
• Fatigue partly due to slowed SERCA & altered Ca²⁺ handling.
• Rigor mortis: ATP depletion means cross-bridges cannot detach; demonstrates energy requirement for relaxation, not contraction.

Formulae & Quantitative Relations

• SERCA stoichiometry: $\text{ATP} + 2\,Ca^{2+}<em>{cyto} \rightarrow 2\,Ca^{2+}</em>{SR} + ADP + P_i$
• Ca²⁺ buffering capacity of SR calsequestrin: binds $\sim 40\,Ca^{2+}$ per molecule, keeping free luminal $[Ca^{2+}]$ moderate.
• Force ∝ $\left( \dfrac{[Ca^{2+}]<em>i}{K</em>{0.5} + [Ca^{2+}]_i} \right)^n$ (Hill-type curve, $n \approx 3–4$).

Summary Cheat-Sheet

• Electrical trigger (AP) → Mechanical work (force) via Ca²⁺ as second messenger.
• T-tubules = AP highways; SR = Ca²⁺ warehouse.
• DHPR senses voltage; RyR releases Ca²⁺; SERCA rescues Ca²⁺.
• Troponin/Tropomyosin decide if actin & myosin may meet.
• ATP fuels (a) myosin power & (b) Ca²⁺ re-uptake; absence of ATP arrests relaxation.
• Contraction persists only while $[Ca^{2+}]_i$ is high; relaxation is an active, ATP-dependent process.