MCB 401 Exam #1

What are cell membranes mostly comprised of?

Lipids and proteins

How are phospholipid names given?

Head group

What is a zwitterionic molecule?

Molecules with positive and negative charges that sum to a net 0 charge

What gives cell membranes their physical properties?

Phospholipid distribution

Width (thickness): fatty acid tail length

Density (packing): head groups

How do phospholipids in the cell membrane move?

Laterally along the leaflet without flipping across leaflet, can rotate/flex minimally

What are examples of types of phospholipids?

Glycerol-based, sphingolipids

What is the structure of cholesterol, and how does this affect its function?

Rigid carbon rings with a small polar head, rings make cholesterol a membrane-stiffening agent, small polar head can weakly interact with water while still allowing for flipping across leaflets

What defines the asymmetry between the internal and external leaflets of the cell membrane?

Same total # of phospholipids but a different distribution of types of phospholipids.

Outer: -choline, sphingo-

Inner: -amine

What is the interior of a vesicle called?

Lumen

What are two transbilayer transporters that work on phospholipids, and how do they function?

Flippase: moves PS in

Floppase: moves PC out

Both use ATP to move their respective phospholipids against a concentration gradient

What are integral proteins?

Proteins that are embedded in leaflet or covalently attached to phospholipids

What are peripheral proteins?

Proteins that non-covalently interact with integral proteins or phospholipids (through weak ionic interactions)

What structure do proteins typically form within the membrane?

Nonpolar α-helices

How does lateral protein diffusion work?

Normally, passive diffusion is extremely slow. By using a “lipid raft”, proteins can “ride along” phospholipid movement, which is significantly faster.

Describe the cell-cell contact method for cell communication and signal transduction.

Connexon protein assemblies form gap junctions, or water-filled channels that share the cytoplasm between two cells, to allow for the transfer of inorganic ions, small molecules (<2 kDa) in a [Ca²⁺]_i, cAMP, or pH-dependent manner.

How are adhering junctions mediated?

Ca²⁺-dependent cadherins - increasing Ca²⁺ concentrations makes the junction stiffer and stronger

How does cadherin mediate intracellular processes?

Cadherin clusters → catenin → actin cytoskeleton

Disrupted adhesion → β-catenin dissociation → nuclear translocation → gene transcription

What are tight junctions, what proteins are involved, and why are they important?

Tight junctions: diffusion barriers - blocks water or ion movement

Proteins involved: claudin, occludin

Intracellular activity: tail ends can interact with actin cytoskeleton (stabilization) or Ser/Thr kinases (signaling)

What intercellular activity does ephrin exemplify?

Ligand present on one cell membrane can signal receptors on another cell membrane

How can cell membranes interact with the extracellular matrix (ECM)?

Integrins: transmembrane receptors that can interact with the ECM

Focal adhesion: clustering of intracellular proteins around receptor that interacts with ECM

Note: the ligand for the receptor can be provided by the ECM

How do chemical signals interact intracellularly?

Lipid soluble: diffuses through cell membrane and interacts with cytoplasmic or nuclear receptors

Others: travel through open channels or secondary messengers

What is the general workflow for intracellular signaling?

Recognition → transduction → transmission → modulation → response → termination

Which side of the GPCR binds the ligand?

Extracellular, N-terminus

Which part of the GPCR interacts with the G protein?

G protein interacts with the loop between segments 5 and 6, hydrophilic region

What are the functions of the three subunits of the G protein?

α subunit hydrolyzes GTP and is responsible for downstream signaling, β and γ subunits are also responsible for downstream signaling (albeit different pathways), and α and γ subunits anchor to the membrane

What does adenylyl cyclase do?

Catalyzes ATP → cAMP

What do the α_s, α_i, α_q, and α_t subunits of the G protein do?

α_s: stimulates adenylyl cyclase

α_i: inhibits adenylyl cyclase

α_q: activates phospholipase C (PLC)

α_t: activates cGMP phosphodiesterase (PDE)

What does cGMP phosphodiesterase (PDE) do?

Catalyzes cGMP → GMP using light as a ligand, where increasing light decreases cGMP

What are the two pathways involving PIP₂?

PIP₂ → PLC-catalyzed transformation to diacylglycerol (DAG) → protein kinase C (PKC)

PIP₂ → PLC-catalyzed transformation to IP₃ → release of Ca²⁺ from ER

Describe simple diffusion of solutes across the cell membrane.

Uncharged, lipid-soluble solutes can travel down their chemical gradient.

What is Fick’s law?

J_x = P_x([X]_o - [X]_i), where J_x is the flux rate and P_x is the permeability coefficient

P_x = (Dβ)/α, where D is the diffusion coefficient, β is the partition coefficient, and α is the membrane width/thickness

What are the two requirements for charged solute transport?

Open pathway, favorable driving force by electrochemical gradient

What term describes the dynamic equilibrium reached by a system when the electrical gradient goes against the chemical gradient?

Steady state

What is the equation to derive the “net” driving force by electrochemical gradients?

∆µ_x = RT ln([X]_i/[X]_o) + z_xF(V_i - V_o), where z_x is the valence

What is another term for V_i - V_o?

Membrane potential V_m

How do you interpret the sign of ∆µ_x?

∆µ_x > 0: movement from inside to outside

∆µ_x < 0: movement from outside to inside

∆µ_x = 0: no net movement

How do you determine the equilibrium potential E_x?

E_x = -RT/z_xF ln([X]_i/[X]_o)

How do you determine the net driving force in volts?

V_m - E_x

What is an easy way to interpret the net driving force?

Ionic flux moves V_m to E_x, i.e., if E_x is less negative than V_m, cations move into the cell and anions leave

What are the three main modes of passive transport?

Pores, channels, carriers

What are pores?

Holes in the cell membrane that are always open

What are porins?

Protein-based channels that allow for transport of small solutes (<5 kDa), water

What are aquaporins?

Small, specialized protein-based channels that only admit water molecules in single-file

What are three key properties of ion channels?

1. Gated (intra- or extracellular)

2. Sensor: membrane voltage, ligands, secondary messenger

3. Selectivity filter (usually in the center of the channel)

How does a carrier work?

One side opens at a time, the solute gets trapped in a “vestibule” intermediate, an inner binding site drives conformational changes

What are three main types of active transport?

Pumps, co-transporters, exchangers

What is the difference between primary and secondary active transport?

Primary: uses ATP hydrolysis

Secondary: uses the movement down one electrochemical gradient to move a different solute againt its electrochemical gradient

What do the α and β subunits of the Na-K pump do?

α subunit: catalytic, hydrolyzes ATP

β subunit: assembles the pump and localizes to the membrane

What is the net effect of the Na-K pump?

Outflow of 3 Na⁺, inflow of 2 K⁺, hydrolysis of 1 ATP

Maintains low intracellular sodium and high intracellular potassium

Net loss of one positive charge per cycle

What is the difference between co-transporters and exchangers?

Both couple the movement of one solute down its electrochemical gradient with the movement of another solute against its electrochemical gradient. Co-transporters are general, while exchangers are a specific type that moves two solutes in opposite directions.

What sign is physiological V_m?

Negative

What physiogloical levels are [Na⁺]_i and [K⁺_i]?

Sodium: low

Potassium: high

What is the function and mechanism of action of ouabain?

Function: Inhibits Na/K pump

Mechanism of action: Blocks pore from extracellular side

How are potassium levels kept from getting too high?

Potassium can move out through ion channels

What ion is responible for making cells excitable?

Na⁺

What magnitude is Ca²⁺’s electrochemical gradient, and how does this affect its movement?

LARGE gradient, causes a rapid burst of movement through voltage/ligand-gated channels

What is the input/output of SERCA, and what purpose does it serve?

2 Ca²⁺ in, 2 H⁺ out per 1 ATP, storage into the ER/SR

What is the input/output of PMCA, and what purpose does it serve?

1 Ca²⁺ out, 1 H⁺ out per 1 ATP, extrusion of calcium through the membrane

How does calmodulin (CaM) regulate PMCA?

Ca²⁺-CaM complex binds to the C-terminus of PMCA, lowers PMCA’s calcium threshold via its catalytic domain, when calcium levels drop, the complex dissociates

What is the input/output of NCX, and in what type of cells is it found?

1 Na⁺ in, 1 Ca²⁺ out, found in neurons and excitable cells

Is water movement active, passive, or both?

ONLY passive, either through membrane diffusion or aquaporins

What single quantity are osmolality and osmolarity based on?

Number of solute particles

In what direction of osmolality does water move?

Low to high osmolality

What is the equation to derive the net “driving force” for water movement?

∆µH₂O = RTV_w(Osm_o - Osm_i) + V_w(P_i - P_o), where V_w is the volume of 1 mol. H₂O, Osm measures osmolarities, and P measures hydrostatic pressures

What is special about P_i - P_o for animal cells?

It is approximately zero

Why is hydrostatic pressure in animal cells essentially zero?

Animal cell membranes are flexible so cannot tolerate large changes in hydrostatic pressure

Describe the Gibbs-Donnan effect.

Negatively-charged large macromolecules (carbohydrates and proteins) create a negative V_m, forcing cations and anions to reach a balance at that V_m (Donnan potential)

What is the Donnan ratio, and what does it mean for it to equal 0.5?

r = [Cation]_o/[Cation]_i = [Anion]_i/[Anion]_o, r = 0.5 corresponds to a point at which all permeable ions are in equilibrium across the membrane

How does the cell regulate NaCl levels to keep from bursting?

Extrusion of Na⁺ via the Na/K pump alongside passive Na/K influx/efflux

What would happen to a cell if the Na/K pump was blocked by ouabain?

Na⁺ cannot be effectively extruded from the cell, so Cl^- levels increase to compensate, the osmololality within the cell increases, and it swells

What are the two “sides” of the epithelial cell, and how are they oriented?

Apical - faces lumen

Basolateral - faces ECF

How are the number of tight junctions related to the levels of paracellular transport?

More tight junctions = less paracellular transport

How is V_m coupled between epithelial cells?

Gap junctions

How do desmosomes provide structural support?

Uses a unique type of cadherin to anchor cells by their intermediate filaments

What features are present on apical brush borders?

Microvilli, membrane-bound enzymes

How is transepithelial voltage calculated?

Voltage difference between apical and basolateral sides → V_bl - V_a

How is the number of tight junctions related to the electrical resistance of an epithelial cell?

More tight junctions = more electrical resistance

How is sodium transported in epithelial cells?

Na⁺ enters on the apical side through ENaC channels and is extruded on the basolateral side by Na/K pumps

How is K⁺ taken in from Na/K pumps extruded in epithelial cells?

Extruded by basolateral channels

How are glucose levels controlled in epithelial cells?

Na/glucose cotransporter on the apical side, extruded basolaterally by GLUT

How are Cl^- levels controlled in epithelial cells?

Brought in by the NKCC on the basolateral side, extruded through apical channels

How do Cl^- levels affect paracellular transport of Na⁺?

Extrusion of Cl^- on the apical side creates a negative charge in the lumen, increasing paracellular transport of Na⁺

How is H₂O permeability across epithelia regulated?

Differential expression of aquaporins, differential membrane lipid composition

How is an H₂O flux generated in the epithelium, even with a low difference in osmolarity between the apical and basolateral sides?

High constitutive aquaporin expression, modest hyperosmolarity in the regions around basolateral membrane infoldings (and between adjacent epithelial cells)