Lecture 5

Membrane Potential and Resting Cellular Environment

• Action Potential Characteristics:

  • Action potentials are rapid, transient electrical events occurring across sub-second thresholds measured in milliseconds ($ms$).

  • An action potential represents a large, rapid shift in the membrane potential of a neuron.

• Ionic Distribution and Rest Membrane Potential:

  • The resting membrane potential (RMP) of a typical neuron ranges between $-\text{60}\,mV$ and $-\text{70}\,mV$.

  • This negative charge inside the neuron relative to the extracellular space is established by the unequal distribution of specific cations, anions, and intracellular proteins.

  • Extracellular Cations:

    • Sodium ($Na^+$): Found in high concentrations outside the cell.

    • Calcium ($Ca^{2+}$): Found in high concentrations outside the cell.

  • Intracellular Cation:

    • Potassium ($K^+$): Found in high concentrations inside the cell.

  • Anions and Intracellular Charge:

    • Chloride ($Cl^-$) is an anion found in higher concentrations in the extracellular space.

    • Despite extracellular $Cl^-$ presence, the inside of the cell remains negatively charged due to an abundance of large, negatively charged intracellular proteins.

    • Intracellular proteins pull the internal charge toward a negative voltage, whereas extracellular cations pull the external charge toward a positive voltage.

• Site of Action Potential Initiation:

  • Action potentials originate at the axon hillock (also termed the initial segment).

  • The axon hillock integrates incoming potentials to determine whether an action potential will be generated.

Electrophysiological Phases and Experimental Observations

• Key Phases of the Action Potential:

  • Threshold: The specific membrane potential voltage that a neuron must reach to trigger an action potential. If the voltage does not reach threshold, no action potential fires.

  • Depolarization: The phase where membrane potential moves toward zero, becoming less negative and less polarized.

  • Repolarization: The phase where the membrane potential moves back down toward negative values, becoming re-polarized.

  • Hyperpolarization: The phase where the membrane potential overshoots the resting membrane potential, becoming more negative than at rest.

  • Mechanistic distinction: The overshoot during hyperpolarization occurs because specific ion channels remain open longer than necessary, rather than as an immediate mechanism to re-establish ion balance. Returning to resting potential from hyperpolarization restores ionic homeostasis.

  • Non-Action Potential Hyperpolarization: Hyperpolarization can occur independently of an action potential. Activation of selective anion channels (such as $Cl^-$ channels) allows $Cl^-$ influx without cation influx, driving the membrane potential below RMP.

• In Vitro Electrophysiology Experiments:

  • Experimental Setup: A neuron placed in a petri dish with a yellow stimulating electrode delivering current/voltage, and two recording electrodes connected to amplifiers placed along the axon:

    • Red recording electrode: Located proximal to the soma.

    • Purple recording electrode: Located distal to the soma, closer to the axon terminal.

  • Hyperpolarizing Stimuli: Injecting step-wise increases of negative voltage produces negative deflections in membrane potential below RMP (hyperpolarization).

  • Subthreshold vs. Suprathreshold Depolarizing Stimuli:

    • Small positive voltage steps induce small positive deflections (subthreshold depolarization).

    • Increasing positive voltage steps yields larger positive deflections.

    • A sufficiently large voltage step pushes the membrane potential to threshold, firing a full action potential.

• Stimulus Intensity vs. Action Potential Amplitude:

  • Increasing stimulus strength beyond threshold does not increase the peak amplitude (height) of the action potential.

  • Instead, stronger stimuli increase the frequency (total number) of action potentials generated over time.

• Unidirectional Current Flow and Temporal Shift:

  • When an action potential is triggered, recordings from the proximal (red) and distal (purple) electrodes reveal a temporal shift: the action potential peak appears later at the distal electrode.

  • Experiments utilizing three recording electrodes positioned sequentially along the axon confirm that current flows strictly in one direction: from the axon hillock toward the axon terminal.

  • Re-recording an action potential at the same electrode from a single stimulus is prevented by the refractory period.

Ion Channel Kinetics During Action Potential Generation

• Leaky Potassium Channels:

  • Also called open potassium ($K^+$) channels.

  • These channels are non-gated and remain continuously open under all physiological conditions.

  • They permit bidirectional movement of $K^+$ ions (inward or outward) across the membrane.

• Voltage-Gated Sodium Channels:

  • Remain closed during resting state.

  • Possess a physical gate located on the extracellular side of the pore.

  • Extracellular Gate Rationale: Because $Na^+$ concentrations are higher extracellularly, ungated channels would allow continuous $Na^+$ influx down its concentration gradient, causing excitotoxicity and seizures.

  • Voltage Sensing Mechanism: Cytoplasm flows directly into the intracellular mouth of the channel. Charged amino acid residues within the channel protein detect voltage changes within the cytoplasm and trigger gate opening upon reaching threshold.

• Threshold and Influx:

  • Across different neurons, threshold potential ranges from $-\text{55}\,mV$ to $-\text{40}\,mV$ (in specific examples, threshold is $-\text{40}\,mV$).

  • When depolarizing currents shift the axon hillock membrane potential to threshold, voltage-gated $Na^+$ channels open rapidly.

  • Driven by both concentration and electrical gradients, $Na^+$ ions flood into the intracellular space, creating a rapid depolarization peak reaching positive potentials (e.g., $+\text{30}\,mV$ to $+\text{40}\,mV$).

• Inactivation States and Refractory Periods:

  • Absolute Refractory Period:

    • Occurs during the rapid depolarization peak.

    • All voltage-gated $Na^+$ channels are fully engaged and open; no additional stimulus, regardless of strength, can fire another action potential.

  • Channel Inactivation States:

    • Inactivated vs. Closed: Inactivated channels have open structural gates but are functional non-operational/blocked, preventing $Na^+$ passage.

    • Open-State Inactivation: Occurs during strong depolarization at the peak and early repolarization.

    • Closed-State Inactivation: Occurs as the membrane transitions through hyperpolarization.

  • Relative Refractory Period:

    • Occurs during repolarization and hyperpolarization.

    • A second action potential can fire, but requires an exceptionally strong depolarizing stimulus.

    • Clinical Context (Seizures): In pathological states such as seizures, excessive excitatory neurotransmission (e.g., glutamate hyperactivation) provides massive depolarizing input, forcing neurons to fire continuously during the relative refractory period.

Repolarization, Hyperpolarization, and Re-establishing Homeostasis

• Voltage-Gated Potassium Channels:

  • Also known as delayed rectifying potassium channels or delayed activating potassium channels.

  • These channels stay closed at rest and open slowly in response to positive membrane depolarization.

  • Unlike leaky $K^+$ channels, delayed rectifying $K^+$ channels permit unidirectional outward flow of $K^+$ ions (efflux).

  • The efflux of positive $K^+$ charges repolarizes the membrane back toward negative potentials.

• Hyperpolarization and The Sodium-Potassium Pump:

  • Slow closure of delayed rectifying $K^+$ channels allows continued $K^+$ efflux, causing the membrane potential to overshoot RMP into hyperpolarization.

  • At hyperpolarization, excess $Na^+$ resides inside the neuron, and excess $K^+$ resides outside.

  • Sodium-Potassium ATPase ($Na^+/K^+$ ATPase Pump):

    • An energy-dependent membrane transport protein.

    • Activated by combination of membrane voltage and ion concentration gradients.

    • Hydrolyzes $1$ molecule of ATP to actively pump $\text{3}\,Na^+$ ions OUT of the cell and $\text{2}\,K^+$ ions IN to the cell.

    • Restores the baseline ion concentration gradients and returns membrane potential to RMP, at which point active pumping ceases.

• Deactivation of Sodium Channels:

  • Following hyperpolarization, voltage-gated $Na^+$ channels transition from the inactivated state to the deactivated state.

  • Deactivated channels are closed but structurally ready to re-open if threshold potential is reached again.

Comprehensive Step-by-Step Cascade of Action Potential Events

• Step 1: Resting State:

  • High extracellular $Na^+$ concentration; high intracellular $K^+$ concentration.

  • RMP sits between $-\text{60}\,mV$ and $-\text{70}\,mV$.

  • Leaky $K^+$ channels are open (bidirectional); voltage-gated $Na^+$ and $K^+$ channels are closed.

• Step 2: Presynaptic Input & Synaptic Potentials:

  • Neurotransmitters released from presynaptic terminals bind to ligand-gated cation ($Na^+$) channels on postsynaptic dendrites or soma.

  • Opening of ligand-gated $Na^+$ channels allows $Na^+$ influx, producing an Excitatory Postsynaptic Potential (EPSP).

• Step 3: Passive Propagation & Integration:

  • Influxed $Na^+$ ions propagate passively down the soma toward the axon hillock.

  • Individual EPSPs are graded; multiple incoming EPSPs from apical and basal dendrites summate spatially and temporally at the axon hillock.

• Step 4: Threshold Activation:

  • If integrated graded potentials shift axon hillock membrane voltage to threshold ($-\text{40}\,mV$), voltage sensors on voltage-gated $Na^+$ channels trigger rapid channel opening.

• Step 5: Depolarization Phase:

  • Massive influx of $Na^+$ rapidly depolarizes the neuron from threshold to peak positive voltage ($+\text{30}\,mV$ to $+\text{40}\,mV$).

• Step 6: Inactivation & Delayed Potassium Opening:

  • At peak depolarization, voltage-gated $Na^+$ channels enter open-state inactivation (Absolute Refractory Period).

  • Delayed rectifying voltage-gated $K^+$ channels open, allowing rapid $K^+$ efflux.

• Step 7: Repolarization & Hyperpolarization:

  • Unidirectional $K^+$ efflux drives membrane potential back negative (Repolarization).

  • Sustained $K^+$ efflux overshoots baseline RMP (Hyperpolarization / Relative Refractory Period).

• Step 8: Resetting Homeostasis:

  • Voltage-gated $K^+$ channels close.

  • Voltage-gated $Na^+$ channels transition to deactivated (closed but responsive) states.

  • $Na^+/K^+$ ATPase pumps $\text{3}\,Na^+$ out for every $\text{2}\,K^+$ brought in, consuming ATP until RMP and physiological ion concentrations are restored.

Axonal Propagation Dynamics and Saltatory Conduction

• Continuous Propagation in Unmyelinated Axons:

  • Unmyelinated axons contain continuous arrays of voltage-gated $Na^+$ channels along the entire axonal membrane.

  • $Na^+$ influx at one segment depolarizes adjacent membrane regions, triggering sequential voltage-gated $Na^+$ channel opening.

  • Drawback: Active propagation across every micrometer of membrane is energy-intensive and extremely slow.

• Passive Propagation:

  • Passage of ionic current through the internal cytoplasm without opening voltage-gated channels.

  • Drawback: Fast over very short distances, but charge rapidly degrades and diffuses over longer distances.

• Myelinated Axons and Saltatory Conduction:

  • Myelin: Lipid insulation wrapped around axon segments, synthesized by oligodendrocytes in the Central Nervous System (CNS).

  • Nodes of Ranvier: Uninsulated gaps along the axon containing concentrated clusters of voltage-gated $Na^+$ and $K^+$ channels.

  • Saltatory Conduction Mechanics:

    • An action potential generated at the axon hillock or a Node of Ranvier causes $Na^+$ influx.

    • Charge propagates passively and rapidly beneath the insulated myelinated segment without decaying significantly.

    • Upon reaching the next Node of Ranvier, the charge depolarizes the node to threshold, triggering active propagation (re-charging the action potential).

    • This alternating combination of passive flow under myelin and active recharging at Nodes of Ranvier dramatically increases signal transmission speed.

Neurotransmitter Release at the Axon Terminal

• Axon Terminal Events:

  • The action potential reaches the terminal membrane of the axon via saltatory conduction.

  • Depolarization opens voltage-gated Calcium ($Ca^{2+}$) channels at the terminal.

  • $Ca^{2+}$ rushes into the intracellular terminal space down its concentration gradient.

  • Intracellular $Ca^{2+}$ influx triggers synaptic vesicles containing neurotransmitters to migrate to, dock, and fuse with the presynaptic terminal membrane.

  • Neurotransmitters are released into the synaptic cleft via exocytosis to transmit chemical signals to the adjacent postsynaptic neuron.

  • Divalent Ionic Specificity: Magnesium ($Mg^{2+}$) cannot replace $Ca^{2+}$ in driving vesicle exocytosis.

Pharmacology, Neurotoxins, and Clinical Pathology

• Voltage-Gated Sodium Channel Blockers:

  • Tetrodotoxin (TTX) (isolated from pufferfish) and Saxitoxin: Specifically block voltage-gated $Na^+$ channels.

  • Consequence: Blockade of $Na^+$ influx prevents action potential generation entirely, leading to paralysis and fatal neurological failure.

• Voltage-Gated Sodium Channel Activators / Inactivation Inhibitors:

  • Batrachotoxin: Holds voltage-gated $Na^+$ channels permanently open, preventing inactivation.

  • Consequence: Continuous, unchecked $Na^+$ influx inundates the neuron with positive charges, causing excitotoxicity and functional paralysis.

• Voltage-Gated Potassium Channel Blockers:

  • Agitoxin and Beta-venerotoxin: Block voltage-gated $K^+$ channels.

  • Consequence: Inhibits outward $K^+$ efflux, preventing repolarization and arresting the neuron in a permanently depolarized state.

• Antidote/Antagonist Dynamics:

  • Counteracting channel-blocking neurotoxins requires high-affinity competitive antagonists capable of displacing the toxin from its specific binding site on the channel protein.

• Multiple Sclerosis (MS):

  • Autoimmune neurodegenerative disease characterized by progressive destruction and breakdown of myelin sheaths (demyelination) in the CNS.

  • Pathophysiological Mechanism: Demyelination disrupts insulated passive charge propagation along axons. Current leaks across uninsulated membrane regions, preventing voltage signals from reaching threshold at Nodes of Ranvier.

  • Clinical Impact: Drastic slowing or total failure of action potential conduction, leading to progressive degradation of motor, sensory, muscular, and cognitive functions.