cell membranes and signalling

Movement of molecules across cell membranes

Diffusion; movement of molecules from one location to another as a result of their random thermal motion

• Molecules eventually are distributed uniformly within a container

• Solutes are redistributed from regions of high concentration

• Equilibrium will eventually be reached

At time A

• Glucose is at 20mmol/L in section 1

• Glucose is at 0mmol/L in section 2

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At time B

• Some molecules that have moved into section 2 will move back into section 1

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At time C

• Diffusion equilibrium is reached

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Flux; amount of material crossing a surface in a unit of time

Net flux; the difference between two one-way fluxes

This means three different fluxes can be identified; each one way flux, then the net flux

Net flux is most important as it reflects movement of material from one compartment to another

Net flux always proceeds from regions of higher concentration to regions of lower concentration

Magnitude of flux always depends on

• Temperature

  • Higher temp means faster movement of molecules

  • Greater flux

• Mass of the molecule

  • Molecules with more mass are slower so have smaller net flux

• Surface area

  • More surface area between 2 regions means more space for diffusion so larger net flux

• Medium through which molecules are moving

  • e.g molecules move quicker through air

Distance limits diffusion

Diffusion through lipid bilayers and protein channels

Molecules diffuse through membranes at a speed x1000 to x1000000 times slower than they would through an equally thick layer of water

Some nonpolar molecules diffuse rapidly through lipid portions of membranes

• Oxygen

• Carbon dioxide

• Fatty acids

• Steroid hormones

Lipophilic substances move through easily

Hydrophilic/polar molecules do  not diffuse readily through membranes

Electrical forces and ion movement

Ions (Na+, K+, Cl-, Ca2+) use specific protein channels to diffuse in and out of cells

• Channels; integral proteins spanning the lipid bilayer

• Several proteins may aggregate, and each form a subunit of the wall of the channel

• Specificity is based on

  • Pore size

  • Charge

  • Binding sites

Regulation of diffusion through ion channels

• Protein channels are regulated

• This controls the movement of ions into and out of a cell

  • Ligand gated

    • Specific molecule (ligands/chemical messengers) binds to channel

    • Leads to allosteric/covalent change in protein shape

  • Voltage gated

    • Change in membrane potential

    • Movement of charged areas

    • Changes protein shape

  • Mechanically gated

    • Physically deforms protein

    • Conformational change

Mediated transport systems

Used for molecules that are too large/charged to enter the cell

Protein transporters bring molecules in/out of cell by conformational changes

Factors determining magnitude of solute flux

• Saturation of transport binding sites

• Number of transporters in the membrane

• Rate at which conformational change occurs

• Facilitated diffusion

  • Net flux of a molecule across a membrane from a higher concentration to a lower concentration until concentration of solute is equal on each side

• Active transport

  • Uses energy to move molecules against concentration gradient

    • Pumps can become saturated

  • Primary

    • Uses ATP for energy

      • 1 ATP associates with a transporter

      • ATPase removes a phosphate, phosphorylating the transporter

        • Covalent modulation

      • Leads to conformational change in the molecule, increases affinity of solute binding site

    • Na+/K+ ATPase is a primary active transporter in every cell

    • Helps establish and maintain membrane potential

      • Most cells also have

        • Ca2+ ATPase

        • H+ ATPase

        • H+/K+ ATPase

  • Secondary

    • Uses an electrochemical gradient across a membrane to drive the process

      • Electrical forces of the charges on the ion must be considered

    • Uses electrochemical gradient to transport solutes against concentration gradient

      • Low Na+/high solute inside cell

      • Electrochem gradient directs Na+ into cell

      • Na+ binds to one site, solute binds to another

      • Na+ is released into cell with solute

        • Transporters have two binding sites; one for ion + one for solute

        • Could also have counter transport

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Electrochemical gradients

Membrane potential; separation of electrical charge across a membrane

Electrochemical gradient considers difference in electricity across the membrane

Osmosis

Osmosis: The net diffusion of water across a membrane, which is dependent on water concentration

Osmolarity is the total solute concentration of a solution

• Movement of water across plasma membranes is mediated by aquaporins

• Some membranes are more permeable to water as the nuber/type of aquaporins varies in different membranes

• The number of aquaporins can be altered in response to certsin signals (e.g ADH)

We can label oure water as 55.5M is we do 1000g/18

Membrane above is permeable to both solute and water

Membrane above is impermeable to solute

• The same concentrations of water and solute will be reached at equilibrium, but the volumes of the compartments will HAVE to change as only water can diffuse

Osmotic pressure: the force required to prevent the floe of water into a solution

Ligand: any molecules or ion bound to a protein by one of the following forces

• Electrical attraction between oppoaitely charged ionic or polae groups on the ligand and a protein

• Weaker attractions due to hydrophobic forces between nonpolar regions on the two molecules

  • Should not involve covalent bonds

  • Reversible

A protein can have several specific binding sites for different ligands

• Binding ligands changes the conformation of a protein

• Activates or inhibits function

Chemical specificity; the ability of a binding site to bind specific ligands

• The protein and the ligand must be close enough + complementary to bind

Binding sites that tightly bind a ligand: high-affinity binding sites

Binding sites that weakly bind a ligand: low-affinity binding sites.

Different proteins may be able to bind the same ligand—that is, may have the same chemical specificity—but may have different affinities for that ligand.

Saturation; the fraction of the total binding sites that are occupied at any given time

An equilibrium is rapidly reached between unbound ligands in solution and their corresponding protein-binding sites.

Saturation depends on

• Concentration of unbound ligand in solution

• Affinity of binding site for ligand

Regulation of binding site characteristics

• Changing protein shape; alters binding of ligands

• Regulating protein synthesis and degradation

Change in charge distribution/in polarity of molecules surrounding protein can influence its shape

• Alteration is selective

  • Allosteric modulation

    • A protein has 2 binding sites

    • Binding to one (regulatory) changes the shape of the other (functional

    • Cooperativity: when a ligand binds to the first of several functional sites on a molecule, this induces a change that increases the affinity of other functional sites)

  • Covalent modulation

    • Covalent binding of charged chemical groups to side chains

    • Msot common is phosphorylation by a kinase

    • A phosphotase removes the phosphate group

Enzymes and chemicall energy

Catabolism: breakdown of organic molecules

Anabolism: synthesis of organic moleules

Reaction rate: how many molecules of a product form in a unit of time

Activation energy: energy to overcome the mutual repulsion from electrons surrounding the atoms that need to bump into each other

Law of mass action: the concentration of reactants or products can determine the direction at which the net reaction proceeds

Cofactors

• Trace metals

  • Mg, Fe, Zn, Cu

• Bind to enzyme

• Alters conformation to enzyme binds to substrate

• Allosteric regulation

Coenzyme

• Used to describe an organic molecules

• Participates as a substrate within a reaction

• Vitamin derived

  • NAD+

  • FAD

The three main factors affecting enzyme-mediated reactions are:

1) Substrate concentration

2) Enzyme concentration

3) Enzyme activity

Metabolic pathway; sequence of enzyme mediated reactions leading to the formation of a particular product

Pathways by lipid soluble messengers

• Bind to intracellular receptors

  • Steroid hormones are lipid soluble

    • Glucocorticoids

    • Vitamin D

    • Sex hormones

    • Thyroxine

  • Enter nucleus and act as transcription

• Diffuse through plasma membrane

• Bind to specific DNA sequences

• Slower than membrane receptors, but sustained response

Pathways by water soluble messengers

• Bind to extracellular parts of intrinsic membrane proteins

  • Dopamine, adrenaline, melatonin

• Broad range of receptors

  • Ion channels

  • g-protein associated receptors

  • Ones with intrinsic kinase activity

• Triggers an intracellular signalling cascade

• Can activate downstream mediators

  • Affect transcription

• Faster than lipid/steroid receptors

First messenger

• Chemical receptor that reaches cell and binds to receptors

Secondary messenger

• Enter/are generated in cytoplasm due to receptor activation

  • Diffuse through cell transmitting signal

Protein kinases

• Transfer phosphate to protein from ATP

Types of receptors

Type A: ligand gated ion channels

• Activated by first messenger (ligand)

• Conformational change leads to channel opening

• Increases net diffusion of a specific type of ion for this channel

• Membrane potential changes

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Type B: function as enzymes

• Intrinsic enzyme activity

• Most will just phosphorylate tyrosine

  • This means they are tyrosine kinases

• Messenger binding causes conformational change

• Receptor autophosphorylates its own tyrosine groups

• Phosphotyrosines act as docking sites for cytoplasmic proteins

• Docking proteins activate other proteins for multiple other pathways

  • All pathways have phosphorylation of cytoplasmic proteins

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Type C: interact with cytoplasmic Janus Kinases (JAKs)

• Do not have intrinsic kinase activity, so use a cytoplasmic kinase

• When ligand binds, a conformational change causes activation of the cytoplasmic kinases

• JAK are cytoplasmic kinases

  • All 4 are tyrosine kinases

  • Expressed differently across different tissues

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Type D: G-protein receptors

• g-protein

  • Protein complex on surface of cytosol

  • A family of heterotrimeric proteins

  • Made of 3 subunits

    • Alpha, beta, gamma

  • Alpha subunit binds GDP (OFF) and GTP (ON)

• Binding of ligand causes conformational change in conformation

• Receptor is activated

• Alpha subunit affinity for GTP increases

  • When the alpha subunit binds to GTP, it dissociates from the other 2 subunits

• Alpha subunit links to another plasma membrane protein

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Signalling

G-protein-coupled receptors use downstream signalling

• When receptor binds, the G protein activates adenylyl cyclase

• Adenylyl cyclase Is a membrane enzyme that catalyses the conversion of cytosolic ATP to cAMP

  • Cyclic 3'-5'- adenosine monophosphate

• cAMP acts as a second messenger

  • Binds to + activates cAMP-dependent protein kinase/protein kinase A/PKA

• PKA phosphorylates downstream targets

cAMP is not the only second messenger

• Ca2+ is usually maintained at a very low concentration in the cytosol

• Creates a large electrochemical gradient

• Promotes diffusion of Ca2+ into the cytosol

• If a receptor is stimulated, an ion channel opens, and the Ca2+ levels change

  • Ca2+ is released from the endoplasmic reticulum

• Ca2+ binds to many proteins in the cytosol

  • Calmodulin is an example, and the most important

  • Calmodulin changes shape

  • Active calcium-calmodulin activates/inhibits kinases

    • e.g calmodulin-dependent kinases

  • These activate other proteins

Cessation of signalling

• Signal transduction pathways are eventually shut off

  • Chronic overstimulation of a cell can be detrimental

• Starts with a decrease in the concentration of first messenger molecules

  • Metabolised by enzymes

  • May simply diffuse away

• Receptor may also be inactivated

  • Chemical alteration e.g phosphorylation

    • Lowers affinity for ligand

  • Receptors may be removed and engulfed by endocytosis

Interfering with signalling

• Arachidonic acid is a polyunsaturated fatty acid, derived from phospholipids in plasma

  membrane

• Uses enzyme phospholipase A2 to split from the membrane in response to stimulus in receptor

• Can be metabolised in 2 different pathways

  • Cyclooxygenase and lipooxygenase

  • Can interfere with signalling

    • Aspirin inhibits cyclooxygenase

    • Corticosteroids inhibit phospholipase A2

  • This is due to the production of 4 eicosanoids

    • Can act as intracellular messengers

    • Usually released locally and act in a paracrine or autocrine manner