Cell Biology · Advanced: Membranes & Transport
2.2 Membrane Proteins and Permeability
On this page 5 sections
Why this matters
Membrane proteins are the cell's interface with the world. They constitute roughly 30% of all protein-coding genes in the human genome and are the targets of more than half of all FDA-approved drugs. Every physiological process — nerve conduction, nutrient uptake, hormone signaling, immune surveillance, mitochondrial ATP synthesis — depends on membrane proteins. Their diversity is staggering: they function as channels, transporters, pumps, receptors, adhesion molecules, and enzymes. Understanding how proteins embed in and interact with the lipid bilayer is essential for pharmacology (drug design often targets transmembrane domains), structural biology (membrane proteins are notoriously difficult to crystallize), and disease biology (mutations in membrane proteins cause cystic fibrosis, long QT syndrome, retinitis pigmentosa, and hundreds of other disorders).
The college version
Core Explanation
Membrane Proteins: Classification by Mode of Association
Membrane proteins are operationally defined by how tightly they associate with the bilayer and whether they penetrate the hydrophobic core.
1. Integral (Intrinsic) Membrane Proteins These span the bilayer (transmembrane) or are deeply embedded in one leaflet and cannot be removed without disrupting the membrane with detergents. They are further subdivided:
- Type I (single-pass): N-terminus outside, single TM helix, C-terminus inside. Example: LDL receptor.
- Type II (single-pass, inverted): N-terminus inside, C-terminus outside. Example: transferrin receptor.
- Type III (multi-pass): Multiple TM helices weaving through the bilayer. Example: G protein-coupled receptors (7 TM helices), glucose transporters (12 TM helices).
- Type IV (multi-subunit): Multiple polypeptide chains associate to form a transmembrane complex. Example: the T-cell receptor complex.
2. Peripheral (Extrinsic) Membrane Proteins These do not penetrate the hydrophobic core. They associate with the membrane surface through:
- Electrostatic interactions with lipid head groups (e.g., annexins binding PS in a Ca²⁺-dependent manner).
- Binding to the exposed portions of integral membrane proteins. They can be stripped from the membrane by changes in pH or ionic strength — no detergent required.
3. Lipid-Anchored Proteins Covalently attached to a lipid moiety that inserts into the bilayer:
- GPI-anchored proteins: Attached to glycosylphosphatidylinositol in the exoplasmic leaflet. Examples: alkaline phosphatase, CD55 (decay-accelerating factor).
- Fatty-acylated proteins: Palmitoylation (reversible thioester linkage to cysteine) or myristoylation (amide linkage to N-terminal glycine). These anchor proteins to the cytoplasmic leaflet. Example: Src kinase (myristoylated).
- Prenylated proteins: Farnesyl or geranylgeranyl isoprenoid groups attached to C-terminal cysteine. Example: Ras GTPases.
Structural Solutions for Spanning the Bilayer
The hydrophobic core of the bilayer (~3 nm thick) is a hostile environment for polar polypeptide backbones. Unpaired hydrogen-bond donors and acceptors in a peptide backbone are energetically expensive to bury. Evolution has produced two elegant solutions:
α-Helical Transmembrane Segments The α-helix satisfies all backbone hydrogen bonds internally (i to i+4), leaving no unpaired donors or acceptors. A stretch of ~20–25 hydrophobic amino acids forms a helix long enough to span the core. The side chains face outward into the lipid, so they must be hydrophobic (Leu, Ile, Val, Phe, Ala, etc.). Multi-pass proteins pack several helices together, often creating a central pore lined with polar residues if the protein functions as a channel or transporter. The vast majority of eukaryotic plasma membrane proteins use this architecture.
β-Barrels In β-barrels, the polypeptide backbone forms β-strands that run perpendicular to the membrane plane and wrap into a closed cylinder. All backbone hydrogen bonds are satisfied between adjacent strands, and the outward-facing side chains are hydrophobic. The interior of the barrel can be hydrophilic, forming a pore. β-barrels are found almost exclusively in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts — a reflection of their evolutionary origin from bacterial endosymbionts. Examples: porins (general diffusion pores in bacteria), VDAC (voltage-dependent anion channel in the mitochondrial outer membrane).
Hydrophobic Matching
Transmembrane proteins have a hydrophobic belt that must match the thickness of the bilayer's hydrophobic core. If there is a mismatch:
- Positive mismatch (protein hydrophobic belt thicker than bilayer): The bilayer stretches to match, or the protein tilts, or the protein aggregates to reduce the exposed hydrophobic surface.
- Negative mismatch (protein hydrophobic belt thinner): The bilayer thins locally, also energetically costly.
Hydrophobic matching is thought to contribute to protein sorting: the plasma membrane is thicker (~35 Å hydrophobic core, enriched in cholesterol and sphingolipids) than the ER membrane (~25 Å). Proteins with longer TM domains tend to accumulate in the plasma membrane; those with shorter TM domains are retained in the Golgi or ER. This is not the sole determinant of sorting, but it is a contributing physical mechanism.
Permeability: What Can Cross the Bare Bilayer?
The pure lipid bilayer is an excellent barrier. Permeability coefficients vary over roughly nine orders of magnitude:
| Molecule | Permeability coefficient (cm/s) | Mechanism |
|---|---|---|
| O₂, CO₂, N₂ | ~10⁰ to 10⁻¹ | Freely permeable (small, nonpolar) |
| H₂O | ~10⁻² to 10⁻³ | Slow but measurable (small, polar) |
| Urea | ~10⁻⁶ | Very slow (polar, larger) |
| Glycerol | ~10⁻⁶ to 10⁻⁷ | Very slow |
| Glucose | ~10⁻¹⁰ | Essentially impermeable |
| Na⁺, K⁺, Cl⁻, Ca²⁺ | <10⁻¹² | Effectively impermeable without proteins |
Rules of thumb:
- Small hydrophobic molecules (O₂, CO₂, steroid hormones) dissolve in the hydrocarbon core and diffuse through freely.
- Small uncharged polar molecules (water, urea, glycerol) cross slowly. Water's surprisingly high permeability (relative to its polarity) is due to transient packing defects in the bilayer that create small aqueous pores — and also due to the presence of aquaporins in most cells.
- Large uncharged polar molecules (glucose, sucrose) are effectively impermeable.
- Ions (Na⁺, K⁺, Cl⁻, Ca²⁺) are virtually impermeable. The Born energy — the energetic cost of moving a charged species from water (dielectric constant ~80) into the hydrocarbon core (dielectric constant ~2) — is enormous (tens of kcal/mol). Ions absolutely require channels or transporters.
Electrochemical Gradients
The direction and magnitude of ion movement across a membrane is determined by the electrochemical gradient — the sum of two forces:
1. Chemical (Concentration) Gradient
Ions tend to move from high to low concentration. The free-energy change for moving one mole of ion X from side 1 to side 2 is:
ΔG_conc = RT ln([X]₂ / [X]₁)
where R is the gas constant and T is the absolute temperature.
2. Electrical (Voltage) Gradient
If there is a membrane potential (V_m), moving a charged particle through it involves electrical work. For an ion of charge z:
ΔG_elec = zFV_m
where F is Faraday's constant and V_m is the membrane potential (inside relative to outside).
Combined Electrochemical Driving Force
ΔG_total = RT ln([X]₂ / [X]₁) + zFV_m
At equilibrium (ΔG_total = 0), the Nernst equation gives the equilibrium potential for a single ion:
E_X = (RT / zF) ln([X]_out / [X]_in)
When multiple ions are involved, the Goldman-Hodgkin-Katz (GHK) equation predicts the resting membrane potential by weighting each ion's contribution by its permeability.
Experimental Evidence
- Hydropathy plots (Kyte–Doolittle): Sliding-window analysis of amino-acid hydrophobicity identifies likely TM segments as hydrophobic peaks of ~20 residues. This remains the standard bioinformatic tool for predicting membrane protein topology.
- X-ray crystallography of bacterial reaction centers (Deisenhofer, Michel, Huber; Nobel Prize 1988): The first high-resolution structure of a membrane protein complex, confirming the existence of TM α-helices.
- K⁺ channel structure (MacKinnon, Nobel Prize 2003): Determined the structural basis of ion selectivity — the selectivity filter precisely coordinates dehydrated K⁺ ions.
- Overton's Rule (1899): Charles Overton observed a correlation between a molecule's lipid solubility and its ability to enter cells — the first systematic evidence that the plasma membrane is lipid-based.
Compare/Contrast: Types of Membrane Proteins
| Feature | Integral TM | Peripheral | Lipid-Anchored |
|---|---|---|---|
| Penetrates bilayer core? | Yes | No | No (anchor only) |
| Removal conditions | Detergent | pH, salt, or chelators | Detergent or phospholipase |
| Common secondary structure | α-helices, β-barrels | Globular domains | Globular domain + lipid moiety |
| Examples | GPCRs, ion channels, GLUT1 | Annexins, spectrin | Ras (prenylated), alkaline phosphatase (GPI) |
| Facing | Both leaflets (TM) | One leaflet | One leaflet (anchor-specific) |
Disease and Clinical Relevance
- Cystic fibrosis: The most common mutation (ΔF508) in CFTR, a chloride channel, causes misfolding and ER retention. The protein fails to reach the plasma membrane, disrupting Cl⁻ and water transport across epithelia.
- Long QT syndrome: Mutations in cardiac voltage-gated K⁺ channels (KCNQ1, hERG) delay ventricular repolarization, predisposing to arrhythmias and sudden cardiac death.
- Aquaporin-2 defects: Mutations in the water channel AQP2 cause nephrogenic diabetes insipidus — the collecting duct cannot concentrate urine, leading to massive water loss.
- Retinitis pigmentosa: Mutations in rhodopsin, a 7-TM photoreceptor, cause its misfolding and aggregation, leading to photoreceptor degeneration.
- Paroxysmal nocturnal hemoglobinuria (PNH): Already covered in 2.1, but worth reiterating — GPI-anchor synthesis defect → loss of GPI-anchored complement regulators on erythrocytes.
Common Misconceptions
| Misconception | Reality |
|---|---|
| "Membrane proteins float freely like corks." | Many are tethered to the cytoskeleton, confined by fences, or clustered in nanodomains. |
| "Any hydrophobic helix can span a membrane." | It must be long enough (~20 residues) and the flanking residues influence orientation (positive-inside rule). |
| "Water crosses the membrane easily because it's small." | Water is polar and crosses pure lipid bilayers slowly; most cellular water flux is through aquaporins. |
| "All transport proteins are channels." | Channels and transporters are distinct: channels form pores; transporters undergo conformational changes to shuttle substrates. |
| "The membrane potential only matters for neurons." | All cells have a resting membrane potential; it drives ion movements, nutrient uptake, and pH regulation in every cell. |
Eli explains
The same idea, in plain words
Explain it like I’m 10
Think of the cell membrane as a wall of oil — water and charged things can't get through it. The cell solves this by embedding special protein machines in the oil wall. Some of these are like doors with locks (channels and transporters) that let specific things pass. Some are antennae (receptors) that catch signals from outside and tell the cell what to do. Some are glue (adhesion proteins) that stick cells together.
How do these proteins stay in the oil wall? They have a greasy belt — a stretch of oily (hydrophobic) amino acids that sits comfortably in the wall's oily interior. The parts that stick out into the watery inside or outside of the cell are made of water-friendly amino acids.
Now, what can sneak through the oil wall without help? Only three things: gases (oxygen, carbon dioxide) that dissolve in oil, tiny oily molecules like steroid hormones, and a little bit of water that manages to slip through cracks. Everything else — sugars, salts, amino acids — needs a protein door. And for charged things like sodium and potassium ions, the wall is an absolute fortress. The cell spends a huge amount of energy pumping ions to create unequal concentrations inside vs. outside, which is like charging a battery. That stored energy runs everything from nutrient uptake to nerve impulses.
Key takeaways
- Transmembrane domains are hydrophobic α-helices (~20–25 residues) or β-barrels; both satisfy backbone H-bonds internally.
- The pure bilayer is permeable only to small, hydrophobic molecules; everything else needs protein-mediated transport.
- The electrochemical gradient is the sum of the chemical (concentration) and electrical (voltage) driving forces — never forget the electrical component for ions.
- Hydropathy plots are the go-to computational tool for predicting TM topology.
- Membrane protein misfolding and trafficking defects are major causes of human disease.
- Q1. You discover a novel membrane protein whose sequence contains a stretch of 22 consecutive hydrophobic amino acids flanked by positively charged residues on the N-terminal side. Predict its topology. What is the "positive-inside rule"?
- Answer
- The 22-residue hydrophobic stretch is almost certainly a transmembrane α-helix. The "positive-inside rule" (von Heijne, 1986) states that positively charged residues (Lys, Arg) are more abundant on the cytoplasmic side of transmembrane helices. The arrangement of flanking charges influences orientation during membrane insertion. Based on this rule, the positively charged N-terminal flank would tend to remain on the cytoplasmic side, meaning the N-terminus faces the cytoplasm and the C-terminus faces the exoplasmic space — consistent with a Type II (N_in, C_out) topology.
- Q2. A cell has the following ion concentrations: [K⁺]_in = 140 mM, [K⁺]_out = 5 mM; membrane potential V_m = −70 mV (inside negative). Is K⁺ at equilibrium? If not, in which direction is the net driving force?
- Answer
- First, calculate the K⁺ equilibrium potential using the Nernst equation (at 37°C, RT/F ≈ 26.7 mV):
- E_K = 26.7 × ln(5/140) = 26.7 × ln(0.0357) = 26.7 × (−3.33) ≈ −89 mV
- Since the actual membrane potential (−70 mV) is more positive than E_K (−89 mV), K⁺ is NOT at equilibrium. The driving force is V_m − E_K = −70 − (−89) = +19 mV. A positive driving force for a cation means the electrical force pulling K⁺ inward (inside is negative) is partially opposed by the concentration gradient pushing K⁺ outward. At −70 mV, the electrical gradient is not strong enough to balance the concentration gradient, so the net driving force pushes K⁺ out of the cell. This is why K⁺ leaks out through open K⁺ channels at rest.
- Q3. Compare and contrast integral and peripheral membrane proteins in terms of (a) how they associate with the membrane, (b) what is required to remove them, and (c) give one example of each.
- Answer
- (a) Integral proteins have one or more hydrophobic transmembrane segments (α-helices or β-barrels) embedded in the bilayer core, making extensive hydrophobic contacts with lipid acyl chains. Peripheral proteins associate via electrostatic interactions with lipid head groups or by binding to exposed surfaces of integral proteins — they do not enter the hydrophobic core.
- (b) Integral proteins require detergents (e.g., SDS, Triton X-100) to disrupt the bilayer and solubilize the protein. Peripheral proteins can be stripped by high salt, alkaline pH, or divalent-cation chelators (e.g., EDTA for Ca²⁺-dependent annexins).
- (c) Integral example: GLUT1 glucose transporter (12 TM helices). Peripheral example: Spectrin, which links to the cortical actin cytoskeleton and binds indirectly to the membrane via ankyrin and band 3.
Study toolsYou’ll learn to
You’ll learn to
- By the end of this topic, you should be able to:
- Classify membrane proteins into integral, peripheral, and lipid-anchored categories, describing the molecular features that determine each class.
- Explain why α-helical transmembrane segments and β-barrels are the two dominant structural solutions for spanning the hydrophobic core.
- Predict the relative permeability of a molecule through a pure lipid bilayer based on its size, polarity, and charge.
- Define an electrochemical gradient and calculate its two components — the chemical (concentration) gradient and the electrical (voltage) gradient.
- Describe hydrophobic matching and explain how it influences protein sorting along the secretory pathway.
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