MEMBRANES

Overview of membranes.

The cell is not an amorphous sack of components, but a complex structure filled with organelles. Examples include:

1. endoplasmic reticulum
2. mitochondria
3. nucleus

Membranes are not passive barriers.

1. They control the structures and environments of the compartments they define, and thereby the metabolism of these compartments.
2. The membrane itself is a metabolic compartment with unique functions.

Membranes are dynamic.

1. They can move.
2. Their components are continuously synthesized and degraded.
3. The primary event in cell death (e.g. myocardial infarction) may be damage to the cell membrane, leading ultimately to cell death.

Preview: In the following sections we will look at membranes from the perspectives of

1. chemical components
2. structure
3. function

The chemical components of membranes

General composition.

1. The components
   • Lipid -- cholesterol, phospholipid and sphingolipid
   • Proteins
   • Carbohydrate -- as glycoprotein
2. Differences in composition among membranes (e.g. myelin vs. inner mitochondrial membrane)
   • Illustrate the variability of membrane structure.
   • This is due to the differences in function. Example:

     Mitochondrial inner membrane has high amounts of functional electron transport system proteins.

     Plasma membrane, with fewer functions (mainly ion transport), has less protein.

   • Membranes with similar function (i.e. from the same organelle) are similar across species lines, but membranes with different function (i.e. from different organelles) may differ strikingly within a species.

Distribution of lipids in membranes

1. There can be large membrane to membrane differences in lipids (compare phosphoglycerides and cholesterol in plasma membrane vs. inner mitochondrial membrane).
2. There can be large differences within classes of lipids (compare the cardiolipin of the inner mitochondrial membrane to other membranes).
3. There are also patterns of differences among the fatty acyl groups of the lipids of various membranes

The reasons for these variations are not known.

The proteins of membranes.

1. Classification of membrane proteins is operational.
   • Definition of "operational classification": based upon how a thing responds to a specified treatment (or operation), rather than upon the intrinsic nature of the thing.
   • There are two kinds of membrane proteins

     Extrinsic (or peripheral): These may be removed from the membrane, or solubilized, by mild treatment, such as shaking with a dilute salt solution. Extrinsic proteins are often thought of as being loosely associated with the membrane surface.

     Intrinsic (or integral): These cannot be removed from the membrane without treatment that destroys the membrane structure, such as dissolving it with detergent. Intrinsic proteins are often pictured as being deeply imbedded in the membrane or transfixing it.

     The biological activity of some proteins depends on whether or not they are associated with membrane.
2. Roles of membrane proteins.
   • Catalytic: enzymes
   • Receptors for signals such as hormones. Binding of the hormone to the protein transmits the signal.
   • Transport
   • Structural. The biconcave discoidal shape of the erythrocyte is due in part to the membrane protein.

Carbohydrates of membranes are present attached to protein or lipid as glycoprotein or glycolipid.

1. Typical sugars in glycoproteins and glycolipids include glucose, galactose, mannose, fucose and the N-acetylated sugars like N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid (sialic acid).
2. Membrane sugars seem to be involved in identification and recognition.

Membrane structure

The amphipathic properties of the phosphoglycerides and sphingolipids are due to their structures.

1. The hydrophilic head bears electric charges contributed by the phosphate and by some of the bases.
   • These charges are responsible for the hydrophilicity.
   • Note that no lipid bears a positive charge. They are all negative or neutral. Thus membranes are negatively charged.
2. The long hydrocarbon chains of the acyl groups are hydrophobic, and tend to exclude water.
3. Phospholipids in an aqueous medium spontaneously aggregate into orderly arrays.
   • Micelles: orderly arrays of molecular dimensions. Note the hydrophilic heads oriented outward, and the hydrophobic acyl groups oriented inward. Micelles are important in lipid digestion; in the intestine they assist the body in assimilating lipids.
   • Lipid bilayers can also form.
   • Liposomes are structures related to micelles, but they are bilayers, with an internal compartment. Thus there are three regions associated with liposomes:

     The exterior

     The membrane itself

     The inside. Liposomes can be made with specific substances dissolved in the interior compartment. These may serve as modes of delivery of these substances.

4. The properties of phospholipids determine the kinds of movement they can undergo in a bilayer.
   • Modes of movement that maintain the hydrophilic head in contact with the aqueous surroundings and the acyl groups in the interior are permitted.

     Rotation

     Lateral diffusion

     Flexing of the acyl chains

   • Transverse movement from side to side of the bilayer (flip-flop) is relatively slow, and is not considered to occur significantly.

Membranes are currently pictured according to the fluid mosaic model.

1. A lipid bilayer composed of phospholipid and cholesterol
2. Proteins. Integral proteins are shown; peripheral proteins may be loosely attached to the surface.

The difficulty with which flip-flop movement of membrane components occurs relates to the sidedness of membranes. Membrane surfaces have asymmetry -- different characteristics on the two sides.

1. There are differences in lipid composition between the sides of a membrane. The mechanism for generating this sidedness is unknown.
2. Membranes also show sidedness with respect to protein composition
   • Different catalytic proteins (enzymes) appear on the two sides of membranes.
   • Carbohydrate is mostly on the outer surface of cell membranes. It is typically attached to the portion of membrane proteins that sticks out.
3. The erythrocyte membrane provides a good model of membrane sidedness.
   • Some proteins (ankyrin, spectrin) are associated with the inner surface of the membrane.
   • Other proteins transfix the membrane (glycophorin), or loop back and forth from side to side (band 3 protein). Note that there is carbohydrate on the exterior portion of glycophorin and band 3.

Membrane fluidity -- according to the fluid mosaic model, proteins and lipids diffuse in the membrane.

Membranes separate and maintain the chemical environments of the two sides of the membrane.

Introduction: there are ion gradients across the mammalian plasma membrane. Here is a comparison of the mean concentration of selected ions outside and inside a typical mammalian cell, giving the ion, the concentration in the extracellular fluid, the intracellular fluid and the difference betwen the two.

 Na+  140 mM   10 mM  14-fold

 K+  4 mM   140 mM  35-fold

 Ca++  2.5 mM   0.1 microM 25,000-fold

 Cl-  100 mM   4 mM  25-fold

Cell membranes maintain these gradients by

• preventing ion flux
• active transport of ions from side to side of the plasma membrane.

Some substances can cross membranes by passive (simple) diffusion.

1. Types of molecules that can cross membranes by diffusion:
   • Water and small lipophilic organic compounds can cross.
   • Large molecules (e.g. proteins) and charged compounds do not cross.
2. Direction relative to the concentration gradient: movement is DOWN the concentration gradient ONLY (higher concentration to lower concentration).
3. Rate of diffusion depends on
   • charge on the molecule -- electric charge prevents movement.
   • size -- smaller molecules move faster than larger molecules.
   • lipid solubility -- more highly lipid-soluble molecules move faster.
   • the concentration gradient -- the greater the concentration difference across the membrane, the faster the diffusion.
4. Direction relative to the membrane: molecules may cross the membrane in either direction, depending only on the direction of the gradient.

Protein channels transport specific ions.

1. Ion channels exist for Na⁺, K⁺ and Ca⁺⁺ movement. These channels are specific for a given ionic species.
2. Channels consist of protein, which forms a gate that opens and closes under the control of the membrane potential.
3. Ion movement through channels is always down the concentration gradient.

Transport of molecules across membranes by carriers (mediated transport).

1. A carrier must be able to perform four functions in order to transport a substance.
   • Recognition -- to specifically bind the substance that is to be transported.
   • Translocation -- movement from one side of the membrane to the other.
   • Release -- on the other side of the membrane
   • Recovery -- return of the carrier to its original condition so it can go through another cycle of transport.
2. Terminology: Carriers are also variously called "porters,""porting systems,""translocases,""transport systems" and "pumps."
3. Carriers resemble enzymes in some of their properties.
   • They are NOT enzymes, as they do NOT catalyze chemical reactions.
   • They are enzyme-like in the following ways.

     They are specific.

     They have dissociation constants for the transported substances which are analogous to Km of enzymes.

     Transport can be inhibited by specific inhibitors.

     They exhibit saturation, like enzymes do. Diffusion, in contrast, is not saturable, and its rate increases with increasing concentration.

4. A general model for transport is that the carrier is a protein which changes conformation during the transport process.
5. Sometimes carriers move more than one molecule simultaneously. Nomenclature:
   • Uniport: a single molecule moves in one direction.
   • Symport: two molecules move simultaneously in the same direction.
   • Antiport: Two molecules move simultaneously in opposite directions.

Passive mediated transport, or facilitated diffusion.

1. The characteristics of a carrier operating by passive mediated transport.
   • Faster than simple diffusion
   • Movement is down the concentration gradient only (like diffusion)
   • No energy input is required -- the necessary energy is supplied by the gradient.
   • The carrier exhibits

     specificity for the structure of the transported substance

     saturation kinetics

     specific inhibitability

2. Examples of passive mediated transport.
   • Glucose transport in many cells.

     A uniport system

     Can be demonstrated by the fact that adding substances with structures that resemble the structure of glucose can inhibit glucose transport specifically.

     It is specific for glucose. The K_m for glucose is 6.2 mM (a value in the neighborhood of the blood concentration of glucose, 5.5 mM) The K_m for fructose is 2000 mM

     The transport process involves attachment of glucose outside the cell. Conformational change of the carrier protein. Release of the glucose inside the cell. There is no need to change K_m for glucose, since the glucose concentration in the cell is very low.

   • Chloride-bicarbonate transport in the erythrocyte membrane. This is catalyzed by the band 3 protein seen previously.

     An antiport system: both ions MUST move in opposite directions simultaneously. The system is reversible, and can work in either direction. Movement is driven by the concentration gradient.

Active mediated transport involves transport against a concentration gradient, and requires energy.

1. There are two sources of energy for active transport.
   • ATP hydrolysis may be used directly.
   • The energy of the Na⁺ gradient may be used in a symport mechanism. The energy of the Na⁺ going down its gradient drives the movement of the other substance. But since the Na⁺ gradient is maintained by ATP hydrolysis, ATP is the indirect source of energy for this process.
2. The characteristics of a carrier operating by active transport.
   • Can move substances against (up) a concentration gradient.
   • Requires energy.
   • Is unidirectional
   • The carrier exhibits

     specificity for the structure of the transported substance

     saturation kinetics

     specific inhibitability

3. How can the substance be released from the carrier into a higher concentration than the concentration at which it bound in the first place?
   • The affinity of the translocase for the substance must decrease, presumably by a conformational change of the translocase.
   • This process may require energy in the form of ATP.
4. Examples of active mediated transport.
   • Ca⁺⁺ transport is a uniport system, using ATP hydrolysis to drive the Ca⁺⁺ movement. There are two Ca⁺⁺ translocases of importance.
     • In the sarcoplasmic reticulum, important in muscle contraction.
     • A different enzyme with similar activity in the plasma membrane.
   • The Na⁺-K⁺ pump (or Na⁺-K⁺ ATPase).
     • An antiport system.
     • Importance: present in the plasma membrane of every cell, where its role is to maintain the Na⁺ and K⁺ gradients.
     • Stoichiometry: 3 Na⁺ are moved out of the cell and 2 K+ are moved in for every ATP hydrolyzed.
     • Specificity: Absolutely specific for Na⁺, but it can substitute for the K⁺.
     • The structure of the Na⁺-K⁺ pump is a tetramer of two types of subunits, alpha₂beta₂. The beta-subunit is a glycoprotein, with the carbohydrate on the external surface of the membrane.
     • The Na⁺-K⁺ ATPase is specifically inhibited by the ouabain, a cardiotonic steroid. Ouabain sensitivity is, in fact, a specific marker for the Na⁺-K⁺ ATPase.
     • The proposed mechanism of the Na⁺-K⁺ ATPase shows the role of ATP in effecting the conformational change.
       • Na⁺ attaches on the inside of the cell membrane.
       • The protein conformation changes due to phosphorylation of the protein by ATP, and the affinity of the protein for Na⁺ decreases.
       • Na⁺ leaves.
       • K⁺ from the outside binds.
       • K⁺ dephosphorylates the enzyme.
       • The conformation now returns to the original state.
       • K⁺ now dissociates.
   • Na⁺ linked glucose transport is found in intestinal mucosal cells. It is a symport system; glucose is transported against its gradient by Na⁺ flowing down its gradient. Both are transported into the cell from the intestinal lumen. Na⁺ is required; one Na⁺ is carried with each glucose. The Na⁺ gradient is essential; it is maintained by the Na⁺-K⁺ ATPase.
   • Na⁺ linked transport of amino acids, also found in intestinal mucosal cells, works similarly. There are at least six enzymes of different specificity that employ this mechanism. Their specificity is as follows.

     Short neutral amino acids: ala, ser, thr.

     Long or aromatic neutral amino acids: phe, tyr, met, val, leu, ile.

     Basic amino acids and cystine: lys, arg, cys-cys.

     Acidic amino acids: glu, asp

     Imino acids: pro and hypro

     Beta-amino acids: beta-alanine, taurine.

Membrane receptors

Cell-cell communication is by chemical messenger.

1. There are four types of signals.
   • Nerve transmission
   • Hormone release
   • Muscle contraction
   • Growth stimulation
2. There are four types of messenger molecules.
   • steroids
   • small organic molecules
   • peptides
   • proteins
3. The messenger may interact with the cell in either of two ways.
   • Entry into the cell by diffusion through the cell membrane (the steroid hormones do this).
   • Large molecules or charged ones bind to a receptor on the plasma membrane.
4. The events associated with communication via these molecules may include the following.
   • Primary interaction of the messenger with the cell (binding by a receptor).
   • A secondary event, formation of a second messenger. (this is not always found).
   • The cellular response (some metabolic event).
   • Termination (removal of the second messenger).

Messenger molecules which diffuse into the cell -- example: steroid hormones.

1. Steroids are lipid soluble, and can diffuse through the plasma membrane.
2. Cells which are sensitive to steroid hormones have specific receptor proteins in the cytosol or nucleus which bind the steroid.
3. The receptor-hormone complex then somehow causes changes in the cell's metabolism, typically by affecting transcription or translation.
4. The mechanism of termination is unclear, but involves breakdown of the hormone.

Plasma membrane receptors.

1. Membrane receptors bind specific messenger molecules on the exterior surface of the cell. Either of two types of response may occur.
   • Direct response: binding to the receptor directly causes the cellular response to the messenger.
   • Second messenger involvement: Binding to the receptor modifies it, leading to production of a second messenger, a molecule that causes the effect.
   • In each case messenger binding induces a conformational change in the receptor protein. Binding of the messenger resembles binding of a substrate to an enzyme in that there is

     a dissociation constant

     inhibition (by antagonists) which may be competitive, noncompetitive, etc.

2. A variety of messengers can bind to various tissues.
   • Various cellular responses may occur, depending on the tissue.
   • Either positive or negative responses may occur, even in the same tissue, depending on the type of receptor.
3. The response of a cell to a messenger depends on the number of receptors occupied.
   • A typical cell may have about 1000 receptors.
   • Only a small fraction (10%)of the receptors need to be occupied to get a large (50%) response.
   • Receptors may have a dissociation constant of about 10 exp -11; this is the concentration of messenger at which they are 50% saturated. Thus very low concentrations of messengers may give a large response.

The acetylcholine receptor of nervous tissue exemplifies a direct response type of receptor.

1. The receptor is a complex pentameric protein which forms a channel through the membrane.
2. Mechanism of action.
   • Binding of acetylcholine, a small molecule, at the exterior surface causes the channel to open. (Binding)
   • Na⁺ and K⁺ flow through the channel, depolarizing the membrane. (Response)
   • The esterase activity of the receptor then hydrolyses the acetylcholine, releasing acetate and choline, and terminating the effect. (Recovery)
   • The process can now be repeated.

Some receptors involve second messengers.

Sometimes the binding of an effector to a receptor leads to the formation of an intracellular molecule which mediates the response of the effector.

1. Definition: This intracellular mediator is called a second messenger.
2. Effect of second messenger formation: Since a receptor usually forms many molecules of second messenger after being stimulated by one molecule of the original effector, second messenger formation is a means of amplifying the original signal.
3. The formation and removal of the second messenger can be controlled and modulated.

Cyclic AMP (cAMP) is a second messenger that mediates many cellular responses.

1. Structure of cAMP: an internal (cyclic) 3', 5'-phosphodiester of adenylic acid.
2. The mechanism of action of cAMP is to activate an inactive protein kinase.
   • Animated activation sequence.
   • Since an active protein kinase which acts on many molecules of its substrate is produced, this process is an amplification of the original signal.
   • Since the protein kinase is activated by cAMP it is called protein kinase A.

3. cAMP is synthesized by the enzyme, adenyl cyclase.
   • The reaction ATP < -> cAMP + PP_i is reversible, but subsequent hydrolysis of the PP_i

     PP_i + H₂O -> 2 P_i

     draws the reaction forward and prevents reversal.

   • Adenyl cyclase is an enzyme, and therefore it is also part of the amplification system.
   • cAMP is degraded by cAMP phosphodiesterase. cAMP + H₂O -> AMP
4. Adenyl cyclase is controlled by two membrane protein complexes, G_s and G_i.
   • G-proteins are a class of proteins that are so named because they can react with GTP. There are G-proteins in addition to the ones under consideration here.
   • G_s and G_i are so named because they stimulate and inhibit, respectively, adenyl cyclase.
5. The action of the G-proteins.
   • Structure: G-proteins are complexes of three different subunits, alpha, beta and gamma. Beta and gamma are similar in the G_s and G_i proteins. The alpha-subunits are different, and are called alpha_s and alpha_i, respectively.
   • Mechanism: Receptor-messenger interaction stimulates binding of GTP to the alpha-subunits. The alpha-subunit with its bound GTP then dissociates from the beta-gamma complex. The alpha-subunit with its bound GTP then acts on adenyl cyclase. alpha_s-GTP stimulates adenyl cyclase. alpha_i-GTP inhibits adenyl cyclase.
6. Termination of the signal occurs at several levels.
   • The alpha-subunit of the G-protein has GTPase activity. After it cleaves the GTP it reassociates with the beta-gamma complex to form the original trimer.
   • cAMP already formed is cleaved by cAMP phosphodiesterase.
   • The hormone gradually and spontaneously dissociates from the receptor.

Inositol triphosphate (IP₃) and diacylglycerol (DG) are also second messengers.

1. Animated activation sequence.
2. IP₃ and DG are synthesized by the enzyme, phospholipase C, which has phosphatidylinositol 4,5-bisphosphate (PIP₂) phosphodiesterase activity. PIP₂ is a normal minor component of the inner surface of the plasma membrane.
3. The phosphodiesterase is controlled by a G-protein in the membrane, which activates the phosphodiesterase.
4. Mechanism: IP₃ and DG have separate effects.
   • IP₃ releases Ca⁺⁺ from the endoplasmic reticulum. The Ca⁺⁺ then activates certain intracellular protein kinases.
   • DG activates protein kinase c, a specific protein of the plasma membrane.
   • Note that both IP₃ and DG activate protein kinases, which in turn phosphorylate and affect the activities of other proteins.
5. Termination of the signal occurs at several levels.
   • IP₃ is hydrolyzed.
   • Ca⁺⁺ is returned to the endoplasmic reticulum or pumped out of the cell.
   • The GTPase activity of the G-protein hydrolyses the GTP, terminating the activity of the phospholipase C.
6. Many systems respond to changes on IP₃ and DG. Be aware of the large number of systems affected.

Insulin and growth factor receptors.

The insulin receptor exemplifies receptors for which no second messenger has yet been identified.

Structure: The insulin receptor is a tetramer with two kinds of subunits, alpha and beta. Disulfide bridges bind them together.

The mechanism of signal transmission is unclear.

Many of the cellular responses are well known, e.g.

1. Glucose transport
2. Protein phosphorylation -- Insulin and many growth factors activate a protein kinase which phosphorylates a tyrosyl residue in the target proteins, including the receptor itself.
   • The phosphorylation of tyrosyl residues is unusual; usually seryl or threonyl residues become phosphorylated.
   • The significance of this type of phosphorylation is unknown.

Termination of the insulin and certain growth factor signals involves internalization and degradation of the hormone within the cell.

1. The receptor-insulin complex migrates to a region of the plasma membrane with the protein clathrin coating its inner surface.
2. This region forms a "coated pit," a region that invaginates and pinches off, forming an intracellular "coated vesicle."
3. The coated vesicle fuses with a lysosome; the lysosomal proteases degrade the hormone specifically, leaving the clathrin and the receptor unharmed.
4. The receptor and clathrin recycle, and are returned to the plasma membrane.

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Last modified 1/5/95