Membrane Transport & Bioenergetics

1. Membrane Transport & Bioenergetics

Thermodynamics, Classification, and Electrophysiology of Solute Movement Across Membranes

1.1 The Thermodynamics of Membrane Transport

Biological membranes are selectively permeable barriers that regulate the intracellular environment. The movement of solutes across these boundaries is governed by fundamental thermodynamic laws. Solutes are broadly classified into uncharged molecules (e.g., urea, glucose, oxygen) and charged ions (e.g., Na⁺, K⁺, Cl⁻, glutamate).

1.1.1 Uncharged Solutes: Chemical Potential Gradient

For uncharged substances, the sole driving force for transport is the concentration gradient across the membrane (the chemical potential difference). The free-energy change (ΔG) associated with transporting one mole of an uncharged solute from an initial compartment with concentration C₁ to a second compartment with concentration C₂ is:

ΔG = RT ln (C₂ / C₁)

ΔG — Gibbs free-energy change (cal/mol or J/mol)

R — gas constant (1.987 cal·K⁻¹·mol⁻¹ or 8.314 J·K⁻¹·mol⁻¹)

T — absolute temperature in Kelvin (K = °C + 273.15)

C₁ — solute concentration in the starting compartment

C₂ — solute concentration in the destination compartment

Figure: Passive vs. active movement of an uncharged solute. When a solute moves from high to low concentration (C₂ < C₁), the ratio C₂/C₁ is below 1, ln(C₂/C₁) is negative, and ΔG is negative — a spontaneous, exergonic process requiring no metabolic energy. Moving the solute up its gradient (C₂ > C₁) makes the ratio and the logarithm positive, so ΔG is positive: an endergonic process that must be coupled to an energy source such as ATP hydrolysis.

1.1.2 Charged Solutes: The Electrochemical Potential

For charged solutes (ions), transport is influenced by two distinct forces: the chemical potential difference, driven by the concentration gradient (RT ln(C₂/C₁)), and the electrical potential difference, driven by the ion's charge interacting with the electric field across the membrane (ZFΔψ). Their sum is the electrochemical potential:

ΔG = RT ln (C₂ / C₁) + ZFΔψ

Z — valency of the solute (e.g. +1 for Na⁺, +2 for Ca²⁺, −1 for Cl⁻ or glutamate)

F — Faraday constant (96,485 C/mol or 23,062 cal·V⁻¹·mol⁻¹)

Δψ — transmembrane potential (V), defined as ψ_inside − ψ_outside

Resting Δψ in eukaryotic cells: −50 mV to −100 mV (cytosolic face negative)

Figure: Electrochemical vector analysis at the membrane. With the cytosolic face negative relative to the outside, a cation whose concentration is higher outside experiences both chemical and electrical forces pulling it inward, so the net driving force is strongly inward. An anion under the same concentration asymmetry has an inward chemical force but an outward electrical force (repelled by the negative interior) — its net force is the vector sum of the two, which can point either way depending on magnitude.

1.1.3 Case Study: Energetics of Glutamate Co-Transport

Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system. To terminate synaptic transmission and prevent excitotoxicity, astrocytes and neurons must clear glutamate from the synaptic cleft against an extremely steep electrochemical gradient, via secondary active transport coupled to the inward transport of sodium ions down their electrochemical gradient.

Problem: analyse the active transport of glutamate from an extracellular concentration of 0.1 mM to an intracellular concentration of 20 mM at physiological conditions (T = 37°C = 310 K, Δψ = −70 mV = −0.07 V). At pH 7.0, glutamate carries a net charge of Z = −1 (pK_a ≈ 2.19 and 4.25 for the two carboxyls; pK_a ≈ 9.67 for the amino group).

Step 1 — free energy required for glutamate influx:

ΔG_Glu = RT ln([Glu⁻]_in/[Glu⁻]_out) + Z_GluFΔψ

Substitute the given values: R = 1.987 cal·K⁻¹·mol⁻¹; T = 310 K; [Glu⁻]_in = 20 mM; [Glu⁻]_out = 0.1 mM; Z_Glu = −1; F = 23,062 cal·V⁻¹·mol⁻¹; Δψ = −0.07 V.
ΔG_Glu = (1.987×310) × ln(20/0.1) + (−1)(23,062)(−0.07)
Evaluate the chemical term: 1.987×310 = 615.97; ln(200) ≈ 5.2983, giving 615.97 × 5.2983 ≈ 3,263.6 cal/mol.
Evaluate the electrical term: (−1)(23,062)(−0.07) = 1,614.3 cal/mol.
Sum the two terms:
ΔG_Glu ≈ 3,263.6 + 1,614.3 ≈ 4,878 cal/mol ≈ 4.9 kcal/mol
Standard textbook rounding sometimes evaluates the chemical term at 3,286 cal/mol, giving a total of 4,900 cal/mol (also ≈ 4.9 kcal/mol).

Step 2 — evaluating the coupling mechanism: the uphill transport of glutamate must be coupled to the downhill influx of Na⁺, which releases roughly −3.3 kcal/mol per mole under physiological conditions. For the coupled system to be thermodynamically favorable, the total net free energy must be negative:

ΔG_net = ΔG_Glu + n(ΔG_Na) < 0

4.9 + n(−3.3) < 0 ⇒ n × 3.3 > 4.9 ⇒ n > 1.48

Conclusion: since stoichiometry must be a whole number, at least two Na⁺ ions must co-transport with each glutamate molecule. Physiologically, the glial glutamate transporter EAAT2 couples the influx of 3 Na⁺ and 1 H⁺, together with the efflux of 1 K⁺, per transport cycle — achieving an even steeper concentration gradient than the minimum required.

1.2 Classification of Membrane Transport Systems

Transport across cellular membranes is systematically categorized by its energy requirements, the structural nature of the transport machinery, and the direction of solute movement.

Figure: Classification of membrane transport systems. Passive transport (no energy input) splits into non-selective simple diffusion through the bilayer and protein-mediated facilitated diffusion, which is further split into slower, saturable carrier-mediated transport and fast, non-saturable channel-mediated transport. Active transport (energy-requiring) splits into primary active transport, which hydrolyses ATP directly (P-, V-, F-type ATPases and ABC transporters), and secondary active transport, which is powered by pre-existing ion gradients and is further divided into symport (co-transport in the same direction) and antiport (exchange in opposite directions).

1.2.1 Passive Transport Mechanisms

Mechanism	Basis	Kinetics / examples
Simple diffusion	Non-polar, hydrophobic passage of small solutes directly through the lipid bilayer, with no membrane protein involved.	Non-selective, non-saturable; depends on the solute's partition coefficient (hydrophobicity). E.g. O₂, CO₂, ethanol.
Facilitated diffusion	Protein-mediated passive transport; solutes move down their electrochemical gradient through specific membrane proteins.	Highly selective, saturable kinetics.
Carrier-mediated (transporters/permeases)	Solute binds a specific site; the protein undergoes a conformational change exposing the site to the opposite side.	Relatively slow (≈10²–10⁴ molecules/s); saturable, hyperbolic kinetics. E.g. GLUT transporters.
Channel-mediated (channels)	Open, water-filled pores spanning the bilayer through which specific ions or water diffuse.	Very fast (≈10⁵–10⁸ ions/s); non-saturable; no rate-limiting conformational cycle per ion.

1.2.2 Active Transport Mechanisms

Mechanism	Basis	Examples
Primary active transport	Transmembrane proteins directly hydrolyse ATP to pump solutes against their electrochemical gradients.	P-type ATPases (Na⁺–K⁺ pump, Ca²⁺-ATPase), V-type proton pumps, ABC transporters.
Secondary active transport	Couples "uphill" transport of a solute against its gradient with "downhill" transport of a driving ion (usually Na⁺ or H⁺) down a gradient established elsewhere by a primary pump.	Glutamate–Na⁺ co-transport (see 1.1.3).
• Symport (co-transport)	Coupled solutes move in the same direction across the membrane.	SGLT-1 sodium–glucose cotransporter.
• Antiport (exchange)	Coupled solutes move in opposite directions across the membrane.	Na⁺–H⁺ exchanger, Na⁺–Ca²⁺ exchanger.

1.3 The Biophysics of Membrane Potential

The membrane potential (Δψ or V_m) is the electrical potential difference across the plasma membrane. It is established by an asymmetric distribution of specific ions across the membrane combined with the selective permeability of the bilayer to those ions.

1.3.1 Establishing Membrane Potential: Chamber Gedankenexperiments

Consider a synthetic system of two compartments, Left (L) and Right (R), separated by a membrane, each loaded with KCl at the stated concentrations:

System A (equiconcentration): both sides contain 1 M KCl and the membrane is selectively permeable to K⁺. With no concentration gradient there is no net chemical driving force, so no net flux of K⁺ occurs and no electrical potential is generated.
System B (asymmetric concentration, K⁺-selective): Left is 1 M KCl, Right is 0.1 M KCl, and the membrane passes only K⁺. K⁺ diffuses down its gradient from Left to Right; because Cl⁻ is trapped on the Left, charge separates — Left becomes net negative, Right net positive. The resulting electric field opposes further K⁺ movement until the electrical force exactly balances the chemical force: the equilibrium potential (E_K) for K⁺.
System C (asymmetric concentration, impermeable): same 1 M / 0.1 M asymmetry, but the membrane is impermeable to both ions. No ion movement, no charge separation, and no membrane potential.
System D (asymmetric concentration, non-selective): same asymmetry, but the membrane is freely permeable to both K⁺ and Cl⁻. Both ions diffuse down their gradients together; concentrations equalise over time, and because positive and negative charge move together, no permanent charge separation — and so no potential — is maintained at steady state.

Key principle: a membrane potential requires two things simultaneously — an asymmetric ion distribution and selective permeability to at least one of the ion species. Remove either condition (Systems A, C, D) and no sustained potential can form; System B, with both conditions present, is the only one that generates an equilibrium potential.

1.3.2 The Nernst Equation

The Nernst equation quantitatively describes the equilibrium potential (E_ion) of a single ion species across a selectively permeable membrane when the chemical and electrical forces are in balance:

E_ion = (RT/ZF) ln ([Ion]_out / [Ion]_in)

Converting to base-10 logarithm and evaluating the constant at physiological temperature (37°C = 310 K) for a univalent cation (Z = +1):

E_ion = 2.303(RT/ZF) log₁₀([Ion]_out/[Ion]_in)

E_ion ≈ 61.5 × log₁₀([Ion]_out/[Ion]_in) (mV, at 37°C)

At room temperature (20°C = 293 K) the constant is instead ≈ 58.2 mV.

Worked example — potassium equilibrium potential in skeletal muscle at 37°C:

[K⁺]_out = 4 mM

[K⁺]_in = 128 mM

R = 8.314 J·K⁻¹·mol⁻¹

F = 9.6485×10⁴ C·mol⁻¹, Z = +1

Evaluate the constant: 2.303×8.314×310 / 96,485 ≈ 0.06154 V.
E_K = 0.06154 × log₁₀(4/128) = 0.06154 × log₁₀(0.03125)
Since log₁₀(0.03125) ≈ −1.5051:
E_K = 0.06154 × (−1.5051) ≈ −0.0926 V = −92.6 mV

When the membrane is permeable only to potassium, the inside of the cell must be negative by 92.6 mV relative to the outside to halt net potassium efflux — this is the potassium equilibrium potential.

1.3.3 The Goldman–Hodgkin–Katz (GHK) Equation

The Nernst equation is restricted to systems with a single permeant ion. Real membranes are simultaneously permeable to multiple ions — principally Na⁺, K⁺, and Cl⁻. The GHK voltage equation gives the actual steady-state membrane potential (V_m or Ψ), weighting each ion's concentration gradient by its relative membrane permeability (P):

Ψ = (RT/F) ln P_K[K⁺]_out + P_Na[Na⁺]_out + P_Cl[Cl⁻]_in P_K[K⁺]_in + P_Na[Na⁺]_in + P_Cl[Cl⁻]_out

Chloride inversion: because Cl⁻ is a negatively charged anion (Z = −1), its concentration terms are inverted relative to the cations — intracellular Cl⁻ appears in the numerator and extracellular Cl⁻ in the denominator.
Permeability weighting: each ion's contribution to Ψ is directly weighted by its permeability coefficient P_i, which is proportional to the number of open ion-selective channels for that ion.
Collapse to Nernst: if the membrane is highly permeable to one ion (e.g. P_K extremely high, P_Na and P_Cl negligible), the GHK equation mathematically collapses into the Nernst equation for that single ion, driving Ψ toward that ion's equilibrium potential.

2. Resting Membrane Potential in Excitable Cells

Animal cells fall into two broad categories: non-excitable cells (e.g. epithelial cells, adipocytes), which maintain a stable, non-fluctuating resting potential, and excitable cells (e.g. neurons, skeletal/cardiac muscle fibers, certain endocrine cells), which maintain a stable resting potential but can rapidly and transiently alter it — generating action potentials — in response to a stimulus.

2.1 Ionic Composition and Permeability Parameters

The resting membrane potential (V_m) of a typical mammalian neuron is approximately −70 mV, established by two interacting factors:

Ion gradients from active transport: the Na⁺–K⁺ ATPase pump continuously expels 3 Na⁺ and imports 2 K⁺ per cycle, maintaining high intracellular K⁺ and low intracellular Na⁺.
Selective leak permeability: the membrane has a high density of constitutively open, non-gated K⁺ leak channels, while resting membranes contain very few open Na⁺ or Cl⁻ channels.

Ion	[C]_out	[C]_in	Nernst potential (E_ion)	Relative permeability (P)
Na⁺	150 mM	15 mM	+61.5 mV	1 (baseline)
K⁺	5 mM	150 mM	−90.2 mV	25–30

Because P_K at rest is 25–30× greater than P_Na, the constant efflux of K⁺ through leak channels dominates the resting electrical state. The exit of positive charge leaves fixed organic anions (proteins, phosphates) behind in the cytosol, charging the inner leaflet negatively. Consequently, the resting potential (−70 mV) sits close to E_K (−90 mV) and far from E_Na (+61.5 mV).

3. The Molecular Biophysics of the Action Potential

An action potential is a rapid, transient, self-propagating reversal of membrane potential polarity in an excitable cell, driven by the opening and closing of voltage-gated ion channels.

Figure: The five phases of the action potential. (1) Resting: membrane sits near −70 mV, with only K⁺ leak channels open. (2) Rising: a stimulus depolarises the membrane to threshold, opening voltage-gated Na⁺ channels and driving a regenerative Na⁺ influx (the Hodgkin cycle) that overshoots to about +30 mV. (3) Falling: Na⁺ channels inactivate while delayed-rectifier K⁺ channels open, driving K⁺ efflux and repolarisation. (4) After-hyperpolarisation: lingering K⁺ permeability drives the potential briefly toward E_K (≈−90 mV). (5) Recovery: delayed-rectifier channels close and leak/pump activity restores the resting potential.

Phase	Na⁺ channel state	K⁺ channel state	Net ion flux
1. Resting	Closed / capable	Closed	Small K⁺ efflux (leak)
2. Rising	Open (activated)	Closed	Massive Na⁺ influx
3. Falling	Inactivated (closed)	Open (activated)	Massive K⁺ efflux
4. Hyperpolarisation	Recovering to closed	Open (closing slowly)	Continued K⁺ efflux
5. Recovery	Closed / capable	Closed	Leak / pump restores gradients

3.1 Conformations of the Voltage-Gated Sodium Channel

The voltage-gated Na⁺ channel (Na_V) has two structural gates that together yield three functional conformations:

Closed but capable (resting state): the voltage-sensitive activation gate is closed, blocking the pore; the cytosolic inactivation gate is open. No ions pass.
Open / activated state: depolarisation to threshold moves the voltage-sensing S4 segments, opening the activation gate. With both gates open, Na⁺ floods in down its electrochemical gradient.
Inactivated (refractory) state: within a fraction of a millisecond, the cytoplasmic inactivation loop (the "ball-and-chain") swings into the pore's inner mouth and plugs it. Even with the activation gate still open, the channel is functionally blocked and cannot reopen until the membrane repolarises and resets the gates.

Figure: Na_V gating cycle. The channel cycles Closed/Capable → Open/Activated → Inactivated → Closed/Resetting → back to Closed/Capable, in step with the phases of the action potential above.

3.2 Step-by-Step Electrophysiological Phases

Resting phase: membrane polarized at −70 mV; high resting P_K via leak channels; voltage-gated Na⁺ and K⁺ channels closed.
Threshold and rising (depolarisation) phase: a local stimulus depolarises the membrane. If it reaches threshold (typically −50 to −55 mV), voltage-gated Na⁺ channels open rapidly, raising P_Na; Na⁺ influx depolarises the membrane further, triggering a positive-feedback loop — the Hodgkin cycle (depolarisation → Na_V opening → Na⁺ influx → further depolarisation) — that overshoots 0 mV to peak near +30 mV (close to E_Na).
Falling (repolarisation) phase: at the peak, Na⁺ channels inactivate (halting influx) while delayed-rectifier K⁺ channels open fully; K⁺ efflux rapidly repolarises the membrane back toward rest.
After-hyperpolarisation phase: because delayed-rectifier K⁺ channels close slowly, elevated P_K persists briefly past −70 mV, driving the potential closer to E_K (≈−90 mV) before K⁺ leak channels and the Na⁺–K⁺ ATPase restore rest.

3.3 Refractory Periods

The refractory period is a brief window during and after an action potential when a cell cannot fire again, or needs a much larger stimulus to do so.

Period	Temporal span	Molecular mechanism	Physiological significance
Absolute refractory	Rising phase and most of the falling phase	Na_V channels are open or inactivated; inactivated channels are locked and cannot reopen for any stimulus.	Makes propagation strictly unidirectional and caps maximum firing frequency.
Relative refractory	After-hyperpolarisation phase	Some Na_V channels have recovered to closed/capable, but open delayed-rectifier K⁺ channels keep the membrane hyperpolarised and conductance high.	A larger-than-normal stimulus can still trigger a full action potential.

All-or-none principle: a sub-threshold stimulus produces no action potential and decays quickly; a supra-threshold stimulus produces a full-sized action potential. All action potentials from a given cell have the same amplitude and shape — stimulus intensity is encoded by firing frequency, not amplitude.

3.4 Graded Potentials vs. Action Potentials

Parameter	Graded potentials	Action potentials
Amplitude	Variable; proportional to stimulus intensity	Constant; independent of stimulus intensity
Duration	Variable (ms to minutes)	Constant (1–2 ms)
Propagation	Passive, electrotonic; decays with distance (decremental)	Active, self-regenerating; no decay over long distances
Threshold	None required	Requires depolarisation to threshold
Refractory period	Absent	Present (absolute and relative)
Summation	Temporal and spatial summation possible	Cannot summate (refractory periods)
Channel types	Ligand- or mechanically-gated	Voltage-gated (Na_V and K_V)
Direction	Multi-directional from origin	Unidirectional (hillock → terminal)

4. Mechanisms of Nerve Impulse Propagation

Once generated at the axon hillock, the action potential must propagate down the axon to its terminal. There are two biological modes of propagation.

4.1 Continuous Conduction (Unmyelinated Axons)

Mechanism: occurs in small, unmyelinated fibers. The inward Na⁺ current during the rising phase depolarises the immediately adjacent patch of membrane, which reaches threshold and opens Na_V channels in the next segment. Speed: 0.5–2.0 m/s. Energy: high — because the entire axonal membrane depolarises, the Na⁺–K⁺ ATPase must continuously restore gradients over the whole surface.

4.2 Saltatory Conduction (Myelinated Axons)

Mechanism: occurs in myelinated fibers wrapped in insulating, lipid-rich myelin (Schwann cells in the PNS, oligodendrocytes in the CNS). The myelin sheath is interrupted every 1–2 mm by unmyelinated Nodes of Ranvier, where Na_V channels are highly concentrated; the membrane under myelin has virtually none. Speed: 15–130 m/s — axial current flows passively through the cytoplasm within insulated internodes, rapidly depolarising the next node to threshold, so the potential appears to "jump" (saltare) node to node. Energy: highly efficient, since depolarisation/repolarisation are restricted to the nodes.

5. Structure and Function of Synapses

A synapse is a specialized junction that allows one neuron to pass an electrical or chemical signal to another cell (another neuron, a muscle cell, or a gland).

5.1 Electrical Synapses

Structure: pre- and postsynaptic membranes are separated by only 2–4 nm and directly linked by gap junctions. Each gap junction is formed by two aligned hemichannels (connexons), each a hexamer of connexin proteins; the aligned connexons form a central pore (1.5–2 nm) — a direct cytoplasmic bridge. Speed: near-instantaneous, no chemical delay. Directionality: bidirectional — current and small second messengers (Ca²⁺, IP₃, cAMP) flow either way. Role: synchronises electrical activity across networks (e.g. cardiac muscle, smooth muscle, some brain circuits).

5.2 Chemical Synapses

Structure: pre- and postsynaptic cells have no direct contact, separated by the fluid-filled synaptic cleft (20–50 nm). Transmission: mediated by neurotransmitters synthesised, packaged into vesicles, and released presynaptically. Synaptic delay: 0.3–2.0 ms, reflecting calcium influx, vesicle exocytosis, diffusion across the cleft, and receptor activation. Directionality: strictly unidirectional, since receptors sit only on the postsynaptic membrane.

6. Detailed Pathway of Chemical Synaptic Transmission

The transfer of information across a chemical synapse is a multi-step biochemical and biophysical cascade.

Action potential arrival depolarises the presynaptic terminal.
Calcium channel activation: depolarisation opens voltage-gated Ca²⁺ channels (N-type or P/Q-type) concentrated in the presynaptic active zone.
Calcium influx: Ca²⁺ rushes in down a steep gradient (extracellular ≈1–2 mM vs. intracellular resting ≈100 nM).
Vesicle docking and exocytosis: the local Ca²⁺ rise is sensed by synaptotagmin, which activates the SNARE complex (v-SNARE synaptobrevin on the vesicle; t-SNAREs syntaxin and SNAP-25 on the presynaptic membrane). This molecular winch pulls the vesicle into the plasma membrane, forcing fusion and releasing neurotransmitter by exocytosis.
Diffusion and receptor binding: neurotransmitter crosses the cleft and binds postsynaptic receptors.
Postsynaptic channel regulation: receptor binding opens or closes specific postsynaptic ion channels.
Postsynaptic potential: the resulting current produces an EPSP (depolarising, toward threshold) or IPSP (hyperpolarising, away from threshold).
Signal termination occurs via enzymatic degradation (e.g. acetylcholinesterase breaking down ACh), reuptake (active transport back into the presynaptic terminal or glia), or simple diffusion out of the cleft.

7. Neurotransmitter Receptors: Ionotropic vs. Metabotropic

Postsynaptic receptors translate chemical neurotransmitter signals into electrical responses, and fall into two fundamental structural classes.

7.1 Ionotropic Receptors (Ligand-Gated Ion Channels)

Structure: multimeric complexes (usually pentamers or tetramers) forming a central water-filled pore, with the neurotransmitter binding site located directly on the channel complex. Mechanism: binding triggers an immediate conformational shift that opens the pore. Kinetics: fast onset (microseconds), short duration (milliseconds).

Receptor	Permeant ion(s)	Effect
Nicotinic ACh (nAChR)	Na⁺ and K⁺ (non-selective cation)	Rapid depolarisation (excitatory)
GABA_A	Cl⁻	Rapid hyperpolarisation (inhibitory)
Glycine receptor	Cl⁻	Rapid hyperpolarisation (inhibitory)
Glutamate (AMPA, NMDA, kainate)	Cations	Fast excitatory transmission

7.2 Metabotropic Receptors (G-Protein Coupled Receptors)

Structure: monomeric, seven-transmembrane-helix (7-TM) proteins with no channel pore. Mechanism: neurotransmitter binds the extracellular face, activating an intracellular heterotrimeric G-protein (αβγ). The dissociated α-GTP and βγ subunits either gate ion channels directly or activate effector enzymes (adenylyl cyclase, phospholipase C) that generate second messengers (cAMP, IP₃, DAG, Ca²⁺), which activate protein kinases that phosphorylate and regulate channels. Kinetics: slower onset (seconds to minutes) but long-lasting, amplifiable, plastic effects.

Examples: muscarinic ACh receptor (mAChR, regulates K⁺ channels), metabotropic glutamate receptors (mGluRs, modulate excitability), GABA_B receptor (increases K⁺ efflux).

8. Physiology of Postsynaptic Potentials: EPSPs vs. IPSPs

Ion currents through activated postsynaptic receptors produce transient, localized changes in the membrane potential of the postsynaptic dendrite or soma.

8.1 Excitatory Postsynaptic Potentials (EPSPs)

Definition: a transient depolarisation shifting the membrane potential closer to threshold. Ionic mechanism: opening of neurotransmitter-gated non-selective cation channels. Both Na⁺ and K⁺ flow through, but at −70 mV the driving force for Na⁺ influx (chemical gradient plus the negative interior both pull it in) vastly exceeds the driving force for K⁺ efflux, so the net current is a massive inward Na⁺ current that depolarises the membrane. Primary transmitters: glutamate, acetylcholine (at nicotinic receptors), aspartate.

8.2 Inhibitory Postsynaptic Potentials (IPSPs)

Definition: a transient hyperpolarisation shifting the potential further from threshold, or a "shunting" effect that stabilises the resting potential. Ionic mechanism: opening of neurotransmitter-gated anion or K⁺ channels.

Channel opened	Ion movement	Effect
Cl⁻ channels	Cl⁻ influx down its gradient	Carries negative charge in, hyperpolarising (e.g. −70→−75 mV)
K⁺ channels	K⁺ efflux (P_K increases)	Hyperpolarises the cell

Primary transmitters: GABA (γ-aminobutyric acid), glycine.

9. Biochemistry of Synaptic Systems: Acetylcholine

Acetylcholine (ACh) is a well-characterised neurotransmitter operating at neuromuscular junctions, autonomic ganglia, and in the central nervous system.

Figure: ACh life cycle. Choline acetyltransferase (ChAT) condenses choline and acetyl-CoA into acetylcholine, which the vesicular ACh transporter (VAChT) packages into vesicles for exocytosis. After receptor activation, acetylcholinesterase (AChE) hydrolyses ACh back to choline and acetate; choline is recycled via a sodium-dependent transporter.

9.1 Synthesis and Packaging

Synthesised in the presynaptic cytoplasm from choline (dietary and recycled) and acetyl-CoA (from mitochondrial glucose metabolism), catalysed by choline acetyltransferase (ChAT):

Choline + Acetyl-CoAChAT→Acetylcholine + CoA

Synthesised ACh is actively pumped into synaptic vesicles by the vesicular acetylcholine transporter (VAChT), powered by an H⁺ gradient established by V-type ATPases.

9.2 Acetylcholine Receptors: nAChR vs. mAChR

Nicotinic (nAChR): an ionotropic pentameric complex (skeletal muscle stoichiometry 2α,β,γ,δ); each of the two α-subunits binds ACh, and the pore (1.5 nm) opens only once both sites are occupied, functioning as a non-selective cation channel. Na⁺ influx dominates, generating an EPSP (an "endplate potential" at the neuromuscular junction) that triggers a muscle action potential.

Muscarinic (mAChR): a metabotropic GPCR. In cardiac fibers, ACh binding to M₂ receptors activates an inhibitory G-protein (G_i); the βγ subunit directly opens inwardly rectifying K⁺ channels, and the resulting K⁺ efflux hyperpolarises pacemaker cells, slowing heart rate.

Pharmacology: atropine, an alkaloid from Atropa belladonna, is a selective competitive antagonist of muscarinic receptors — used clinically to treat bradycardia and to dilate the pupils.

9.3 Signal Termination: Acetylcholinesterase

Acetylcholinesterase (AChE), a highly efficient serine esterase on the postsynaptic membrane and in the cleft, hydrolyses ACh:

Acetylcholine + H₂OAChE→Choline + Acetate

Acetate diffuses away; choline is recycled into the presynaptic terminal by a sodium-dependent choline co-transporter (ChT).

Toxicology: organophosphate insecticides (malathion, parathion) and nerve agents (sarin, VX, soman) bind covalently and irreversibly to AChE's active-site serine. ACh then accumulates continuously, hyperactivating nicotinic and muscarinic receptors body-wide — producing muscle fasciculations followed by depolarisation blockade (diaphragm and skeletal muscle paralysis), salivation, lacrimation, urination, defecation, bronchoconstriction, bradycardia, and death from respiratory failure.

10. Pharmacology of Voltage-Gated Channels and Synapses

Many natural toxins and pharmacological agents act on voltage-gated ion channels or synaptic machinery, providing crucial insights into membrane biophysics.

Toxin	Source	Mechanism	Physiological effect
Tetrodotoxin (TTX)	Pufferfish liver, ovaries, skin (from symbiotic bacteria)	Binds the outer vestibule of Na_V, plugging the pore; blocks all Na⁺ conductance	Prevents action potentials; progressive flaccid paralysis, respiratory failure, death
Saxitoxin (STX)	Marine dinoflagellates (Gonyaulax, Alexandrium); accumulates in shellfish during red tides	Same site as TTX — blocks Na_V pore entry	Paralytic shellfish poisoning: numbness, paralysis, respiratory arrest
Veratridine	Sabadilla plant (Schoenocaulon officinale) seeds	Binds open Na_V, prevents inactivation gate closure — locks channel open	Persistent depolarisation, repetitive firing, muscle spasms
Batrachotoxin (BTX)	Skin secretions of poison-dart frogs (Phyllobates)	Binds open Na_V hydrophobic core, shifts activation threshold negative, blocks inactivation	Channels locked open; cardiac arrhythmia, muscle rigidity, death
Dendrotoxin	Black mamba (Dendroaspis polylepis) venom	Blocks the pore of delayed-rectifier K_V channels	Prevents repolarisation, prolongs the action potential, drives excess Ca²⁺ influx and neurotransmitter release — convulsions, hyperexcitability, death

11. Transport of Macromolecules: Endocytosis and Exocytosis

Macromolecules (proteins, complex carbohydrates, cellular debris) are too large for channels or carriers. Their transport requires vesicular mechanisms that physically remodel the membrane.

11.1 Endocytosis: Internalization Mechanisms

The plasma membrane invaginates around extracellular material and pinches off internally to form a vesicle. Three pathways:

Pinocytosis ("cell drinking"): non-specific internalization of extracellular fluid and dissolved solutes via small, simple invaginations.
Phagocytosis ("cell eating"): non-specific, high-capacity internalization of large solid particles (bacteria, debris), carried out by phagocytes (macrophages, neutrophils). Pseudopodia extend around the particle and fuse, enclosing it in a phagosome that fuses with a lysosome for degradation.
Receptor-mediated endocytosis: a highly selective, high-affinity pathway. Ligands (e.g. LDL, transferrin) bind surface receptors; complexes accumulate in clathrin-coated pits; the clathrin cage drives invagination; the GTPase dynamin severs the vesicle neck; the coat is shed and the vesicle fuses with endosomes to sort cargo.

11.2 Exocytosis: Secretion Mechanisms

Intracellular vesicles fuse with the plasma membrane to release contents (hormones, enzymes, neurotransmitters) or insert new lipids/proteins into the membrane.

Constitutive pathway: continuous, unregulated exocytosis in all eukaryotic cells; vesicles bud continuously from the Golgi and fuse with the plasma membrane, maintaining surface area and secreting matrix proteins.
Regulated pathway: restricted to specialized secretory cells (neurons, endocrine and exocrine cells). Vesicles are stored near the membrane and fuse only in response to a specific signal, most commonly a transient rise in cytosolic Ca²⁺.

12. Synthesis and Integration: The Neuromuscular Junction

To see how these transport mechanisms coordinate in a real physiological system, consider a single muscle contraction triggered at the mammalian neuromuscular junction — requiring the coordinated activity of multiple channels, carriers, and vesicular mechanisms.

Action potential propagation: travels along the motor neuron axon via alternating Na_V opening (depolarisation) and delayed-rectifier K_V opening (repolarisation), propagating rapidly by saltatory conduction across myelinated segments.
Calcium-mediated exocytosis: at the nerve terminal, depolarisation opens voltage-gated Ca²⁺ channels; the resulting influx is sensed by synaptotagmin, which activates SNARE proteins (synaptobrevin, syntaxin, SNAP-25) to drive exocytosis of ACh-containing vesicles.
Ligand-gated channel activation: released ACh diffuses across the cleft and binds nAChRs on the muscle endplate, opening non-selective cation pores and admitting a massive Na⁺ influx that generates an endplate potential (EPSP).
Voltage-gated action potential generation: this local depolarisation reaches threshold in the neighbouring muscle membrane, opening muscle Na_V channels to fire a muscle action potential, which propagates along the fiber and triggers Ca²⁺ release from the sarcoplasmic reticulum to drive contraction.
Signal termination: acetylcholinesterase rapidly hydrolyses ACh into choline and acetate, ending the signal and allowing repolarisation; choline is recycled into the neuron via sodium-dependent co-transporters.

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