PLASMA MEMBRANE AND TRANSPORT

Karp chapter 4, class notes and these notes.

I. Lipids found in membranes.

Phospholipids (phosphoglycerides and sphingomyelins), glycolipids, cholesterol. Structure. Their amphipathic nature.

II. Evidence for the bilayer.

• Stability considerations. Hydrophobic regions of lipid molecules must not be exposed to water.
• Experiment by Gorter and Grendel. Lipids extracted from red blood cells (which have no intracellular membranes) were placed in a Langmuir trough as a monolayer and compressed to reach the liquid state. The area covered by the monolayer was approximately two times the area of the surface of the red blood cells.
• Electron microscopy. In electron micrographs, the cross section of membranes appear as two parallel lines of high electron density separated by a low density region of about 3 nm in thickness, which is the approximate dimension of the hydrophobic region predicted for a bilayer.
• X-ray diffraction studies of myelin. See fig 4.5.
 

III. Membrane proteins.

a. Integral (or intrinsic) and peripheral (or extrinsic). Peripheral proteins are associated to the surface of membrane by ionic and other polar interactions, and can be dissociated using high ionic strength solutions or divalent ion chelators, as EDTA or EGTA. Integral proteins are inserted in the bilayer and, frequently, span the bilayer and have regions of the polypeptide chain exposed to both its cytosolic and to its exoplasmic sides. Whereas peripheral proteins are frequently water soluble, intrinsic ones are not. The hydrophobic regions found within the bilayer induce the formation of multimolecular aggregates that precipitate (hydrophobic interaction or hydrophobic effect). In order to extract these proteins from the membrane and to keep them in suspension, it is necessary to use detergents.

b. Transmembrane proteins. Can be identified by exposing cells to non-penetrating agents that covalently attach a label to certain amino acid residues. The presence of label in an extracted membrane protein indicates that it has regions accessible from the exterior of the cell. Other penetrating labels would indicate accessibility from the cytosolic side. Proteins that are labeled by both types of agents are transmembrane proteins. The regions that span the membrane are most frequently alpha helices about 20 amino acids long with mostly hydrophobic side chains. If the amino acid sequence of a transmembrane protein is known, the number of its transmembrane regions can be quite accurately predicted by constructing a hydrophylicity plot that clearly shows the hydrophobic and hydrophilic regions and their length. Some proteins show transmembrane regions that have beta structure. Bacterial porins are an example: their transmembrane regions are barrel shaped structures formed by multiple beta strands (see Fig 5.3).

The first direct evidence for alpha helical transmembrane regions came from low angle electron microscopy of the membrane of Halobacterium halobium, which contains the protein bacteriorhodopsin (see Fig 4.42). The pigment associated with the protein absorbs light and the complex uses the energy to transport H⁺ to the outside, thereby generating a concentration and electrical gradients that passively drive the H⁺ back into the cell through an ATP synthetase in a manner similar to the way in which ATP is synthesized in mitochondria. The bacterium shows purple patches in its membrane where bacteriorhodopsin is densely packed. This provides enough repetitiveness of the structure to generate an image with sufficient resolution to visualize seven quasi-cylindrical masses traversing the membrane with the dimensions required of alpha helices. Hydrophilicity plots of may other membrane proteins show evidence for seven-pass structures. These proteins include light receptors, odor receptors , receptors for some neurotransmitters and for many hormones. Many other transmembrane proteins show single or multiple pass characteristics. Among them, band III, a 12 or 14 pass anion exchanger found in the red blood cell membrane; glycophorin, a single pass protein also found in the erythrocyte membrane. The first membrane protein for which X-ray diffraction studies rendered structural information at the atomic resolution is the bacterial photosynthetic reaction center of R. viridis, which remains one of the very few for which this kind of structural knowledge exists. It shows eleven transmembrane alpha helices (see Fig 14-17).

IV. The fluid mosaic model of membrane structure.

The notion of the lipid bilayer became well established over 50 years ago, but the role played by proteins in the structure of the membrane remained a matter of discussion for 20 more years. The work of Daniel Branton and associates on freeze fracture and etching (see pg. 780 and fig. 18.15 and fig 18.16 ) in the early 60's provided conclusive evidence about the existence of protein inside the membrane. The ability to label some membrane proteins by applying labels from either side of the membrane also gave strong support to that idea.

A great deal of indirect evidence also pointed to a fluid membrane. The formation of endocytotic and exocytotic vesicles, the phenomena of patching and capping observed with fluorescent antibodies and other similar behaviors strongly supported this idea. Later, a number of ingeniously designed experiments provided very strong and direct evidence. Edidin fused human and mouse cells with the aid of Senday virus. When these hybrid cells were treated with fluorescent anti mouse antibodies immediately after fusion, they showed fluorescence only over the one half of the cell that had the mouse surface antigens, but, after several hours, showed uniformly distributed fluorescence. This indicates that the mouse surface antigens, which are intrinsic membrane proteins, can migrate laterally within the membrane (see pg. 144 and Fig 4.26). Fluorescence recovery after photobleaching (FRAP) experiments provide also direct evidence and quantitative information from which it is possible to calculate the diffusion coefficients of membrane proteins (see pg 145 and Fig 4.27). Similar FRAP measurements done on phospholipids tagged with fluorescent dyes or with spin-labeled compounds show a rapid lateral diffusion for the lipids, but a very slow rate of exchange between monolayers (flip-flop).

a. Factors that influence the degree of fluidity. Phospholipid molecules in artificial bilayers show a diffusion coefficient an order of magnitude larger than those in cell membranes. They also have a much larger range of motion, since plasma membranes seem to have compartments within which lipid molecules move rapidly, but which do not easily exchange them. Membrane proteins also show some limitations to their motility and, in some cases, they are fixed in place by attachments to the cytoskeleton.

Besides this, there are physical and chemical factors that affect the fluidity of the membrane and, with it, the motility of its molecules. Van der Waals interactions between parallel hydrocarbon chains of fatty acids contribute over 1 KJ/mole per CH₂ to the stability of the bilayer. This is a considerable amount of energy and would lead to a rather rigid (solid state) bilayer were it not for the influence of the following:

• Temperature. When a bilayer made up of one kind of phospholipid with a single type of fatty acyl chain is cooled down, it undergoes a change of state that resembles the solidification of a two dimensional liquid: it happens at a given temperature (the freezing point) and with the release of a characteristic amount of heat. If a solidified bilayer is heated, it melts at a given temperature with the absorption of a given amount of heat.
• Length of the fatty acid chains. Shorter chains reduce the stabilty of the bilayer and its tendency to become solid.
• Double bonds in the fatty acid chains. These appear mostly in the cis configuration and, therefore, have a kink in the chain. This interferes with the orderly arrangement of parallel hydrocarbon chains and reduces the interaction between adjacent chains. Consequently, these double bonds contribute to increasing the fluidity of the membrane. This is also the reason why vegetable oils, with long fatty acid chains but abundant unsaturation, are liquid, while solid animal fats, usually with shorter chains but few unsaturated fatty acids, are semisolid.

• Cholesterol. Its molecule strongly interacts with the outer regions of adjacent hydrocarbon chains, but doesn't reach the inner part of the leaflets, in which the chains become separated and much less organized. In mammalian cells, cholesterol contributes to the stability of the bilayer at relatively high temperatures, but interferes with the solidification of the bilayer at low temperatures.
 

V. Special structures in the plasma membrane

• Tight junctions. In epithelial cellular membranes that line certain organs, the tight junctions contribute to separate regions of the membrane that have different membrane proteins and, therefore, different physiological functions (polarized cells). They also contribute to reduce considerably the flow of fluid and solutes across the epithelium. Examples: intestinal lining, walls of renal tubules, pancreatic acinar cells, etc.
• Desmosomes and gap junctions. Create channels of communication between adjacent cells.

B. TRANSPORT

Small hydrophobic molecules diffuse through artifical lipid bilayers with relative ease. Water permeates quite slowly, and the rapid transmembrane movement of water observed in cells must happen through specialized structures. Similarly, ions and hydrophylic and large molecules will not cross lipid bilayers, and also require specialized structures to cross the plasma membrane. These specialized structures are membrane proteins that show different modes of operation and usually show a great deal of specificity for the species transported.

I. Membrane Transport Proteins.

• ATP-powered pumps. Transport specific ions across the membrane against their electrochemical gradient using the energy derived from ATP hydrolysis.
• Ion channels. Allow the passive flow of specific ions down their electrochemical gradient when opened. The ways in which the cells control the opening and closing of channels are varied and depend on the type of channel.

• Transporters. Provide a path for small hydrophobic molecules (sugars, amino acids) and ions to cross the membrane down their concentration or electrochemical gradients. There are three kinds:

• Uniporters. Allow the passage of single molecules down their concentration gradient.
• Symporters. Allow the passage of an ion down its electrochemical gradient only when accompanied by another ion or molecule even against its own gradient
• Antiporters. Allow the passage of an ion down its electrochemical gradient only when another ion or molecule is carried in the opposite direction, even against its gradient.
 

II. Diffusion.

The description of transmembrane diffusion given in class will be sufficient for the kinetics of diffusion. The energetics are simple. It is obvious from Fick's first equation that the distribution of a given solute across a membrane will be at equilibrium (velocity of diffusion is zero) when the concentrations on both sides of the membrane are equal. The delta G for the transport of substance i from side a to side b of the membrane is

Delta G_i /mol = G_i^b /mol- G_i^a/mol= G_i⁰ /mol + RT ln [i]^b - (G_i⁰ /mol + RT ln[i]^a ) = RT ln ([i]^b /[i]^a)

When [i]^b = [i]^a, ln 1= 0 and delta G/mol = 0.       Eq. 1

III. Transport Through Uniporters (Facilitated Transport).

Uniporters do not show preference for a given direction of transport. The glucose transporter in liver cells will equally transport this sugar into the cell as out, the direction being determined by the direction of the concentration gradient. Uniporters show a high degree of specificity either for a single chemical species or for a few related species. For example, the glucose transporter also facilitates the movement of galactose, but will not allow L-glucose to go through. At the same time, these two species show competition for the transporter. If the rate of glucose transport by red blood cells is found to have a certain value in an experiment and, suddenly, galactose is added to the medium, the rate of glucose transport will decrease in proportion to the concentration of added galactose. Galactose, therefore, behaves as a competitive inhibitor of glucose transport. The concentration dependence of the rate of transport is not linear as normal diffusion would be. Instead, it shows the hyperbolic relationship typical of the saturation kinetics seen for the Michaelis-Menten model of enzyme kinetics (see Fig 4.38). This shows that, as for enzymes, the number of transporters is small compared to the number of molecules available for transport, and a high concentration of the latter may saturate the transporting capacity of the system. As with M-M enzymes, these transporters can be characterized by a K_M and a V_MAX.

The properties of these systems were described through experiments performed on intact cells long before techniques to isolate these proteins and to introduce them in artificial liposomes (see Fig 15-4) were available, but experiments that use these more recent techniques yield cleaner and less ambiguous results.

The energetics of facilitated transport are the same as for diffusion.

IV. Ion Channels and Ion Fluxes.

Both the kinetics and the energetics of ionic diffusion need to take into account the effect of the electrical potential difference across the membrane. Although many cells have constantly changing membrane potentials, most show a pretty steady potential. Those that do not will show a stable potential if the internal and/or external influences that determine the changes are removed. This so called resting membrane potential varies between - 30 mV and - 90 mV, depending on the type of cell. The sign is determined by the convention used in measuring it (inside minus outside potential) and by the fact that ionic fluxes determine that cell membranes have a slight excess of negative charges on the cytosolic surface and an equal amount of positive charges on the exoplasmic surface. The existence of this electrical potential difference contributes an additional term to the expression for the delta G of transport from side a to side b of the membrane: zF (V^b - V^a), where z is the ionic charge, F is Faraday's constant (96,500 coulombs per mole) and V is the value of the potential on the corresponding side. The expression for the transport of potassium ions from outside to the inside of a cell is:

Gⁱ /mol- G^o /mol= Delta G/mol = RT ln( [K]ⁱ /[K]^o) + F (Vⁱ - V^o)

where Vⁱ - V^o = E_m is the membrane potential. 

The expression for the transport from inside to outside is:

G^o/mol- Gⁱ /mol= delta G/mol = RT ln ( [K]^o / [K]ⁱ ) + F (V^o - Vⁱ ) = RT ln ( [K]^o / [K]ⁱ - F E_m       Eq 2

The value of R is 8.3144 J K^-1 mole^-1. At 300 K (27 C), RT is 2494 J mole^-1 .

Equations for the kinetics of diffusion identical in form to those derived from Fick's first law of diffusion may be obtained by using the concept of the gradient of chemical potential (or gradient of free energy per mole). Similarly, an electrochemical potential is defined as the delta G per mole for the transport of ions in a mixed concentration and electrical fields, and its gradient is the intensity of the force per mole that drives the diffusion. These ideas about gradients in connection with the kinetics of diffusion will not be further developed here. But the concepts of chemical potential difference and of electrochemical potential difference defined by equations 1 and 2 are the basis for the energetics of diffusion.

Example:

In a cell with an internal [K⁺ ] = 140 mM, an external [K⁺] = 5 mM and a membrane potential Em = - 70 mV, the delta G at 27^o C for inward diffusion of this ion is

Delta G = 2.5 KJ/mol ln (.14/.005) + {96,500 coul/mol · (-.07 V)}/1000J/KJ = 1.6 KJ/mol

which indicates that the inward diffusion is not spontaneous. On the other hand, it shows that the outward diffusion has a delta G = - 1.6 K/mol.

The rate of diffusion (or the magnitude of the diffusional flux) will be expressed as the product of a driving force and a conductance or permeability factor ( J = Force x P). For ions in an electric field, that net force is the sum of a force due to a concentration gradient (Fc) and another due to the electrical potential gradient (Fe). Then, J = (Fc + Fe) P.

a. Membrane potential. The plasma membrane of cells separate two different electrolyte solutions and has different permeabilities for the two main cations present, Na and K. The internal concentrations are 140 mM for K and 10 mM for Na. The external concentrations are 5 mM for K and 150 mM for Na. P_K = 50 P_Na . The equations for the two ionic fluxes are:

J_K = P_K ( Fc_K + Fe) = P_K F_K,Net

J_Na = P_Na (Fc_Na + Fe) = P_Na F            Eq 3

The electrical forces for both ions are equal, since their charges are equal and the membrane potential difference is the same for both. The two concentration forces may be considered to be similar in magnitude, but their directions are opposite. If we select the inward direction as the positive direction, it is clear that the direction of the sodium concentration force is positive while the direction of the potassium concentration force is negative. On the other hand, the direction of the electrical forces are both positive. This means that, if we assume a membrane potential of -70 mV, the net fore acting on sodium ions is the sum of the concentration and electrical forces, while the net force acting on potassium ions is the difference between the concentration and electrical forces. It follows that the net force acting on sodium is larger than the net force acting on potassium, which does not mean necessarily that the sodium flux is larger than the potassium flux, since the value of the respective permeabilities must be considered. At the same time, the condition for attaining a stable membrane potential is that the net electrical current flowing through the membrane be zero. This requires that the two fluxes considered must be equal and opposite

J_K = - J_Na from where P_K /P_Na = - ( F_Na,Net / F_K,Net )

This means that, when the potential has reached a stable value, the ratio of the net forces acting on the ions is equal to the ratio of their permeabilities. The negative signs are just indicating that the direction of the two fluxes and of the two net forces are opposite.

The discussion on active transport of ions, the different classes of transporters, and details about the sodium-potassium pump and the calcium pump found in the muscle cell ER should be learned.

Cotransport (also called by some secondary active transport) systems, mainly those found in the intestinal epithelium and the renal tubular epithelium should be learned.. The energetics of ion transport should be applied to these symporters and antiporters.

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